Non-tumorigenic MDCK cell line for propagating viruses

ABSTRACT

The present invention provides novel MDCK-derived adherent non-tumorigenic cell lines that can be grown in the presence or absence of serum. The cell lines of the present invention are useful for the production of vaccine material (e.g., viruses). More specifically, the cell lines of the present invention are useful for the production of influenza viruses in general and ca/ts influenza viruses in particular. The invention further provides methods and media formulations for the adaptation and cultivation of MDCK cells such that they remain non-tumorigenic. Additionally, the present invention provides methods for the production of vaccine material (e.g., influenza virus) in the novel cell lines of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation and claims benefit under 35 U.S.C.§120 of U.S. patent application Ser. No. 11/304,589, filed Dec. 16,2005, which claims the benefit under 35 U.S.C. §119(e) of the followingU.S. Provisional Application Nos.: 60/638,166 filed Dec. 23, 2004 and60/641,139 filed Jan. 5, 2005, each of which are hereby incorporated byreference herein in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to novel non-tumorigenic MDCK cells, whichcan be used for the production of vaccine material. The non-tumorigenicMDCK cells may be adapted to serum-free culture medium. The presentinvention further relates to media formulations and cultivation methodsfor the propagation of the non-tumorigenic MDCK cells as well as methodsfor maintaining the non-tumorigenic nature of the cell lines of theinvention. The present invention further relates to processes for theproduction of influenza viruses in cell culture using non-tumorigenicMDCK cells. The present invention also relates to the viruses (e.g.,influenza) obtainable by the process described and immunogeniccompositions which contain viruses of this type and/or componentsthereof.

BACKGROUND OF THE INVENTION

Vaccination is the most important public health measure for preventingdisease caused by annual epidemics of influenza. The effective use ofvaccines is dependent on being able to quickly produce large quantitiesof vaccine material (e.g., virus) from a stable and easy to cultivatesource. The rapid development of vaccines and their abundantavailability is critical in combating many human and animal diseases.Delays in producing vaccines and shortfalls in their quantity can causeproblems in addressing outbreaks of disease. For example, recent studiessuggest that there is cause for concern regarding the long lead timesrequired to produce vaccines against pandemic influenza. See, forexample, Wood, J. M., 2001, Philos. Trans. R. Soc. Lond. B. Biol. Sci.,356:1953. Efficient vaccine production requires the growth of largequantities of vaccine material produced in high yields from a hostsystem. Different vaccine materials require different growth conditionsin order to obtain acceptable yields. Vaccine material may be producedin embryonated eggs, primary tissue culture cells, or in establishedcell lines. However, these host systems currently suffer from a numberof limitations detailed below.

Embryonated eggs are typically used for influenza vaccine virusproduction in a time-, labor-, and cost intensive process thatnecessitates the management of chicken breeding and egg fertilization.In addition, influenza vaccine produced in eggs is contraindicated forpersons with egg allergies due to the severe immediate hypersensitivityreaction that can occur. Thus, there has been an effort by the vaccineindustry to develop alternative production platforms that do not utilizeeggs such as producing influenza vaccine in a cell culture system.

The use of primary tissue culture cells is hampered by the difficultiesencountered in developing and maintaining a stable primary cellpopulation. Often established cells lines are used to circumvent thetechnical limitations of primary cells. However, many of these celllines are known to be tumorigenic and as such raise safety concerns andare subject to significant regulatory constraints against their use forvaccine production. In fact, the applicable guidelines of the WorldHealth Organization indicate that only a few cell lines are allowed forvaccine production. Additional problems arise from the use of serumand/or protein additives derived from animal or human sources in cellculture media. For example, variability in the quality and compositionamong lots of additives and the risk of contamination with mycoplasma,viruses, BSE-agents and other infectious agents are well known. Ingeneral, serum or serum-derived substances like albumin, transferrin orinsulin may contain unwanted agents that can contaminate the culture andthe biological products produced from therefrom. Therefore, many groupsare working to develop efficient host systems and cultivation conditionsthat do not require serum or serum derived products.

Consequently, there has been a demand for establishing a non-tumorigeniccell line useful for the production of vaccine materials in a low-cost,highly safe and stable manner preferably in serum-free or in animalprotein-free culture conditions. Such a cell system would beparticularly useful for the production of influenza vaccine material.

Madin Darby Canine Kidney (MDCK) cells have been traditionally used forthe titration of influenza viruses (Zambon M., in Textbook of Influenza,ed Nicholson, Webster and Hay, ch 22, pg 291-313, Blackwell Science(1998)). These cells were established in 1958 from the kidney of anormal male cocker spaniel. The ATCC list the MDCK (CCL 34) line ashaving been deposited by S. Madin and N. B. Darby however, numerousother lineages of MDCK cells are available. Leighton J and his coworkerspublished a series of papers (Leighton et al., 1968, Science 163:472;Leighton et al., 1970, Cancer 26:1022 and Leighton et al., 1971 Europ J.Cancer 8:281) documenting the oncogenic characteristics of the MDCKcells. However, the lineage and passage number of the MDCK cells usedfor these studies was not described and it was already known that MDCKcells from different lineages and different passages showed changes inchromosome numbers and structure (Gaush et al., 1966, Proc. Soc. Exp.Biol. Med., 122: 931) which could result in cells with tumorigenicproperties.

Since one of the major considerations for the acceptability of a cellline for vaccine production concerns the potential malignancy of thosecells the use of MDCK cells for the production of vaccine material usingcurrently described cell lines is limited. Groner et al. (U.S. Pat. No.6,656,720) and Makizumi et al. (U.S. Pat. No. 6,825,036) both purport todisclose cell lines derived from MDCK cells which have been adapted togrow in serum-free media in suspension and which can be utilized for theproduction of influenza virus. However, it has been reported that thereis correlation between the loss of anchorage requirement and thetransformation of normal animal cells to cells which are tumorigenic(Stiles et al., 1976, Cancer Res., 36:3300). Several groups (Kessler etal., 1999, Cell Culture Dev Biol Stand, 98:13; Merten et al., 1999, CellCulture Dev Biol Stand, 98:23 and Tree et al., 2001, Vaccine, 19:3444)purport to describe the use of MDCK cells for the large-scale productionof influenza virus; however, they do not address the potentialtransformation of the MDCK cells used.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention. Inaddition, citation of a patent shall not be construed as an admission ofits validity.

SUMMARY OF THE INVENTION

The present invention provides non-tumorigenic MDCK cells which havebeen adapted to grow in either serum containing or serum-free mediaformulations including animal protein-free (APF) formulations. In oneembodiment, the non-tumorigenic MDCK cells of the invention areadherent. In another embodiment, the non-tumorigenic MDCK cells of theinvention have an epithelial morphology. In yet another embodiment, thenon-tumorigenic MDCK cells of the invention are adherent and have anepithelial morphology. Tumorigenicity is in one embodiment, determinedby the adult nude mouse model (e.g., Stiles et al., 1976, Cancer Res,36:1353, and Example 2 below). Tumorigenicity may also be tested byother assays, for example, by injection into a chick embryo and/ortopical application to the chorioallantois (Leighton et al., 1970,Cancer, 26:1024).

Viruses that can be grown in the MDCK cells of the invention include butare not limited to negative strand RNA viruses, including but notlimited to influenza, RSV, parainfluenza viruses 1, 2 and 3, and humanmetapneumovirus.

The present invention further provides methods and media formulationsuseful for the derivation and maintenance of non-tumorigenic MDCK cells.The MDCK cells of the invention are particularly useful for theproduction of vaccine material such as, for example, viruses.

Other aspects of the invention include methods of producing vaccinematerial (e.g., virus) by culturing any MDCK cell of the invention, in asuitable culture medium under conditions permitting production ofvaccine material and, isolating the material from one or more of thehost cell or the medium in which it is grown.

Immunogenic compositions are also features of the invention. Forexample, immunogenic compositions comprising the vaccine materialproduced as described above and, optionally, an excipient such as apharmaceutically acceptable excipient or one or more pharmaceuticallyacceptable administration component.

Methods of producing immunogenic responses in a subject throughadministration of an effective amount of one or more above describedimmunogenic compositions to a subject are also within the currentinvention. Additionally, methods of prophylactic or therapeutictreatment of a viral infection (e.g., viral influenza) in a subjectthrough administration of one or more above described immunogeniccompositions in an amount effective to produce an immunogenic responseagainst the viral infection are also part of the current invention.Subjects for such treatment can include mammals (e.g., humans).Additionally, such methods can also comprise administration of acomposition of one or more viruses produced in the MDCK cells of theinvention and a pharmaceutically acceptable excipient that isadministered to the subject in an amount effect to prophylactically ortherapeutically treat the viral infection.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures appendix.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Growth of Influenza strains in cells. Panel A is a photographshowing the results of a fluorescent focus assay comparing the spread ofinfection of a representative ca/ts influenza strain in MDCK cells and aVero Cell Clone (27F9). Panel B is a growth curve of influenza strain caA/Vietnam/1203/2004 (H5N1) in MDCK cells. Titers peaked at 48 hours postinfection at ˜8 log₁₀ TCID₅₀/mL and remained stable for the next 3 to 4days.

FIG. 2 outlines the process used for the derivation of MDCK-S PreMCB(passage No. 57). The process is described in detail in Example 2.

FIG. 3 is a photograph showing that MDCK-S cells have an epithelial-likemorphology. The photo was taken 3 days after seeding.

FIG. 4 is the growth curve of MDCK-S cells in 10% FBS DMEM medium. Cellshad about a 1 day lag phase followed by exponential growth enteringstationary phase at day 4 post seeding achieving a maximum density of˜29×10⁶ cells on day 5.

FIG. 5 is a graph of the glucose consumption and lactate production ofMDCK-S cells in 10% FBS DMEM medium. The rates were low during lag phaseincreasing to 2.93 mM/day and 3.43 mM/day for glucose and lactate,respectively.

FIG. 6 is a graph of the glutamine consumption and both glutamate andammonia production of MDCK-S cells in 10% FBS DMEM medium. The glutamineconsumption rate was 0.49 mM/day up to day 4 and the ammonia productionrate was 0.32 mM/day up to day 5. Glutamate did not accumulate in thisstudy.

FIG. 7 is a plot of the distributions of chromosome number in 100metaphase low passage (P61/4) and high passage (P81/24) MDCK-S cells.The chromosome count ranged from 70 to 84 per metaphase with a modalchromosome number of 78 for both the high and low passage cells.

FIG. 8 outlines the process used for the derivation of MDCK-T PreMCB(passage No. 64/5). The process is described in detail in Example 3.

FIG. 9 is a photograph showing that MDCK-T cells have an epithelial-likemorphology. The photo was taken 3 days after seeding.

FIG. 10 is the growth curve of MDCK-T cells in Taub's media. Cells hadno lag phase and were in exponential growth until entering stationaryphase at day 4 post seeding.

FIG. 11 is a graph of the glucose consumption and lactate production ofMDCK-T cells in Taub's media. During the exponential phase the rateswere 1.78 mM/day and 2.88 mM/day for glucose and lactate, respectively.

FIG. 12 is a graph of the glutamine consumption and both glutamateammonia production of MDCK-T cells in Taub's media. The glutamineconsumption rate was 0.36 mM/day up to day 4 and the ammonia productionrate increased linearly up to day 7 at a rate of 0.22 mM/day. Glutamatedid not accumulate in this study.

FIG. 13 is a plot of the distributions of chromosome number in 100metaphase low passage (P61/4) and high passage (P81/24) MDCK-T cells.The chromosome count ranged from 52 to 82 per metaphase for low passagecells and from 54 to 82 for high passage cells.

FIG. 14 is a plot of the distributions of chromosome number in 100metaphase MDCK-T, MDCK-SF101 (passage 71/9) and MDCK-SF102 cells(passage 71/9). Both SF101 and SF102 cells had a modal chromosome numberof 78, with the chromosome count ranging from 70 to 82 and 60 to 80 permetaphase for SF101 and SF102, respectively.

FIG. 15 is a photograph showing that MDCK-SF103 have an have anepithelial-like cell morphology. The photo was taken 3 days afterseeding.

FIG. 16 is the growth curve of MDCK-SF103 cells in MediV SFM103. Cellshad about a 1 day lag phase followed by exponential growth enteringstationary phase at day 4 post seeding achieving a maximum density of˜17×10⁶ cells on day 4.

FIG. 17 is a graph of the glucose consumption and lactate production ofMDCK-SF103 cells in MediV SFM103. During the exponential phase theglucose consumption and lactate production mirrored each other withlactate increasing in concentration as the glucose concentrationdecreased

FIG. 18 is a graph of the glutamine consumption and both ammonia andglutamate production of MDCK-SF103 cells in MediV SFM103. The ammoniaproduction rate increased nearly linearly up to day 7. Glutamate did notaccumulate in this study.

FIG. 19 is a plot of the distributions of chromosome number in 100metaphase MDCK-SF103 cells at passage 87. SF103 cells had a modalchromosome number of 78, with the chromosome count ranging from 66 to80.

FIG. 20 Production Scale Growth and Purification. Panel A is a plot ofthe yield obtained for several vaccine reassortant strains,B/Victoria/504/2000 (˜8 LogTCID 50/mL), A/Sydney/05/97 (˜7.85 LogTCID50/mL) and A/New Calcdonia/20/99 (˜8.2 LogTCID 50/mL), from 250 mLspinner flasks of MDCK-SF103 grown on Cytodex beads. Panel B outlinesone cell culture scale up process which can be utilized for commercialscale production of vaccine material.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery that MDCK cellscan be cultivated under conditions wherein they remain non-tumorigenic.The present invention provides non-tumorigenic cell lines, includingMDCK cell lines and other types of cells which have been adapted to avariety of cell culture conditions including serum-free mediaformulations and are referred to herein as “cells of the invention”. Inaddition, the present invention provides cell culture compositionscomprising cells of the invention and other components including, butnot limited to, media (e.g., a media disclosed herein), mediacomponents, buffers, chemical compounds, additional cell types, viralmaterial (e.g., viral genomes, viral particles) and heterologousproteins. The present invention also provides methods and mediaformulations useful for the cultivation of non-tumorigenic cells,including MDCK cells, with one more specific characteristics includingbut not limited to, being non-tumorigenic (e.g., not forming nodules ina nude mouse) and/or growth as adherent cells and/or having anepithelial-like morphology and/or supporting the replication of variousviruses including but not limited to orthomyxoviruses, paramyxoviruses,rhabdoviruses and flavoviruses. The culture conditions of the presentinvention include serum containing and serum-free media formulations, aswell as animal protein-free (APF) formulations. In addition, the presentinvention also provides methods of producing vaccine material (e.g.,influenza virus) in non-tumorigenic cells, including MDCK cells,preparing vaccine material from non-tumorigenic cells, and methods ofpreventing influenza infection utilizing vaccine materials produced innon-tumorigenic cells. The cells of the invention are particularlyuseful for the production of cold adapted/temperaturesensitive/attenuated (ca/ts/att) influenza strains (e.g., those inFluMist®) which do not replicate as efficiently in other mammalian celllines (e.g., Vero, PerC6, HEK-293, MRC-5 and WI-38 cells).

Cell Characteristics

The cells according to the invention are in one embodiment, vertebratecells. In another embodiment, the cells of the invention are mammaliancells, e.g., from hamsters, cattle, monkeys or dogs, in particularkidney cells or cell lines derived from these. In still anotherembodiment, the cells of the invention are MDCK cells (e.g., derivedfrom ATCC CCL-34 MDCK) and are specifically referred to herein as “MDCKcells of the invention” and are encompassed by the term “cells of theinvention”. In a specific embodiment, the cells of the invention arederived from ATCC CCL-34 MDCK. Cells of the invention may be derivedfrom CCL-34 MDCK cells by methods well known in the art. For example,the CCL-34 MDCK cells may be first passaged a limited number of times ina serum containing media (e.g., Dulbecco's Modified Eagle Medium(DMEM)+10% Fetal Bovine Serum (FBS)+4 mM glutamine+4.5 g/L glucose, orother media described herein) followed by cloning of individual cellsand characterization of the clones. Clones with superior biological andphysiological properties including, but not limited to, doubling times,tumorigenicity profile and viral production, are selected for thegeneration of a master cell bank (MCB). In one aspect, the cells of theinvention are adapted to growth in a media of choice (e.g., a serum-freeor APF media, such as those described herein). Such adaptation may occurprior to, concurrently with, or subsequent to the cloning of individualcells. In certain embodiments, cells of the invention are adapted togrow in MediV SF101, MediV SF102, MediV SF103, MediV SF104 or MediVSF105. Cells of the invention adapted to grow in these media arereferred to herein as “MDCK-SF101, MDCK-SF102, MDCK-SF103, MDCK-SF104and MDCK-SF105” cells, respectively and as “MDCK-SF cells” collectively.In other embodiments, cells of the invention are adapted to grow inserum containing media (e.g., Dulbecco's Modified Eagle Medium(DMEM)+10% Fetal Bovine Serum (FBS)+4 mM glutamine+4.5 g/L glucose),such cells are referred to herein as “MDCK-S” cells. MDCK-SF and MDCK-Scells are also encompassed by the terms “cells of the invention” and“MDCK cells of the invention”.

In a specific embodiment of the invention the cells are of the celllines including, but not limited to, those which have been depositedwith the American Type Culture Collection (10801 University Boulevard,Manassas, Va. 20110-2209) and assigned ATCC Deposit Nos. PTA-6500(Deposited on Jan. 5, 2005), PTA-6501 (Deposited on Jan. 5, 2005),PTA-6502 (Deposited on Jan. 5, 2005), and PTA-6503 (Deposited on Jan. 5,2005), these cells are referred to herein as “MDCK-S, MDCK-SF101,MDCK-SF102 and MDCK-SF103”, respectively and as “the MDCK cells of theinvention” collectively. These deposits will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure. Sincethe strains referred to are being maintained under the terms of theBudapest Treaty, they will be made available to a patent officesignatory to the Budapest Treaty. In one embodiment, the MDCK cells ofthe invention are used to generate a cell bank useful for thepreparation of vaccine material suitable for approval by the U.S. Foodand Drug Administration for human use.

The cells lines MDCK-S, MDCK-SF101, MDCK-SF102, MDCK-SF103, MDCK-SF104and MDCK-SF105 are derived from the cell line MDCK (CCL 34) by passagingand selection with respect to one or more specific characteristicsincluding but not limited to, growing as adherent cells either in serumcontaining, or serum-free media or animal protein-free media, having anepithelial-like morphology, being non-tumorigenic (e.g., not formingnodules in a nude mouse) and/or supporting the replication of variousviruses including but not limited to orthomyxoviruses, paramyxoviruses,rhabdoviruses and flavoviruses.

In one embodiment, the MDCK cells of the invention are non-tumorigenic.Methods for determining if cells are tumorigenic are well known in theart (see, for example, Leighton et al., 1970, Cancer, 26:1024 and Stileset al., 1976, Cancer Res, 36:1353), the method currently preferred bythe U.S. Food and Drug Administration using the nude mouse model isdetailed in Example 2 below. In a specific embodiment, the MDCK cells ofthe invention are non-tumorigenic in the adult nude mouse model (see,Stiles et al., Id and Example 2 below). In another specific embodiment,the MDCK cells of the invention are non-tumorigenic when injected into achick embryo and/or topically applied to the chorioallantois (see,Leighton et al., Id). In still another embodiment, the MDCK cells of theinvention are non-tumorigenic in the adult nude mouse model but not wheninjected into a chick embryo and/or topically applied to thechorioallantois. In yet another embodiment, the MDCK cells of theinvention are non-tumorigenic in the adult nude mouse model and wheninjected into a chick embryo and/or topically applied to thechorioallantois. In still another embodiment, the MDCK cells of theinvention are non-tumorigenic after at least 20 passages, or after atleast 30 passages, or after at least 40 passages, or after at least 50passages, or after at least 60 passages, or after at least 70 passages,or after at least 80 passages, or after at least 90 passages, or afterat least 100 passages in a medium. In yet another specific embodimentthe medium is a media described herein (e.g., Medi SF103).

Tumorigenicity may be quantified in numerous ways known to one of skillin the art. One method commonly utilized is to determine the “TD₅₀”value which is defined as the number of cells required to induce tumorsin 50% of the animals tested (see, e.g., Hill R. The TD₅₀ assay fortumor cells. In: Potten C, Hendry J, editors. Cell clones. London:Churchill Livingstone; 1985. p. 223). In one embodiment, the MDCK cellsof the invention have a TD₅₀ value of between about 10¹⁰ to about 10¹,or between about 10⁸ to about 10³, or between about 10⁷ to about 10⁴. Ina specific embodiment, the MDCK cells of the invention have a TD₅₀ valueof more than about 10¹⁰, or of more than about 10⁹, or of more thanabout 10⁸, or of more than about 10⁷, or of more than about 10⁶, or ofmore than about 10⁵, or of more than about 10⁴, or of more than about10³, or of more than about 10², or of more than about 10¹.

In another embodiment, the non-tumorigenic cells of the invention growas adherent cells either in serum containing or serum-free media oranimal protein-free media. In still another embodiment, thenon-tumorigenic cells of the invention have an epithelial-likemorphology. In yet another embodiment, the MDCK cells of the inventionsupport the replication of various viruses including but not limited toorthomyxoviruses, paramyxoviruses, rhabdoviruses and flavoviruses. It iscontemplated that the MDCK cells of the invention may have anycombination of one or more specific characteristics including but notlimited to, being non-tumorigenic, growing as adherent cells, having anepithelial-like morphology and supporting the replication of variousviruses.

It is contemplated that each and every passage of the MDCK cells of theinvention is documented in sufficient detail such that the completelineage of each cell line is available. The documentation of each andevery passage may facilitate approval by the U.S. Food and DrugAdministration and other regulatory bodies around the world for the useof the MDCK cells of the invention for the preparation of vaccinematerial.

In another embodiment, the MDCK cells of the invention are free ofmicrobial contaminants (e.g., bacterial, viral and fungal contaminants).Methods for testing for the presence of bacterial and fungalcontaminants are well known in the art and routinely performed bycommercial contractors (e.g., BioReliance®, Rockville, Md.). Acceptedmicrobial sterility and mycoplasm tests are detailed in Example 2 below.Specific examples of microbial agents which may be tested for are listedin Table 6.

In yet another embodiment, the MDCK cells of the invention support thereplication of viruses including but not limited to orthomyxoviruses(including influenza A and/or B strains), paramyxoviruses (including RSVA and/or B, human metapneumovirus and parainfluenza 1, 2 and/or 3),rhabdoviruses and flavoviruses. In a specific embodiment, the MDCK cellsof the invention support the replication of cold adapted/temperaturesensitive (ca/ts) influenza viruses such as those found, for example, inFluMist® (Belshe et al., 1998, N Engl J Med 338:1405; Nichol et al.,1999, JAMA 282:137; Jackson et al., 1999, Vaccine, 17:1905) and/orreassortant viruses having these as backbones. One indication of theability of a cell to support viral replication is the yield of virusobtained from an infected cell culture. Viral yield can be determined bynumerous methods known to one skilled in the art. For example, viralyield can be quantified by determining the concentration of viruspresent in a sample according to a median tissue culture infectious dose(TCID₅₀) assay that measures infectious virions. The TCID₅₀ values areoften reported as the log₁₀ TCID₅₀/mL. In one embodiment, the MDCK cellsof the invention support the replication of influenza viruses (e.g.,ca/ts strains) to a log₁₀ TCID₅₀/mL of at least 6.0, or at least 6.2, orat least 6.4, or at least 6.6, or at least 6.8, or at least 7.0, or atleast 7.2, or at least 7.4, or at least 7.6, or at least 7.8, or atleast 8.0, or at least 8.2, or at least 8.4, or at least 8.6, or atleast 8.8, or at least 9.0, or at least 9.2, or at least 9.4, or atleast 9.6, or at least 9.8. In another embodiment, the MDCK cells of theinvention support the replication of influenza viruses (e.g., ca/tsstrains) to a log₁₀ TCID₅₀/mL of at least about 6.0, or at least about6.2, or at least about 6.4, or at least about 6.6, or at least about6.8, or at least about 7.0, or at least about 7.2, or at least about7.4, or at least about 7.6, or at least about 7.8, or at least about8.0, or at least about 8.2, or at least about 8.4, or at least about8.6, or at least about 8.8, or at least about 9.0, or at least about9.2, or at least about 9.4, or at least about 9.6, or at least about9.8.

It will be understood by one of skill in the art that the cells of theinvention will generally be part of a cell culture composition. Thecomponents of a cell culture composition will vary according to thecells and intended use. For example, for cultivation purposes a cellculture composition may comprise cells of the invention and a suitablemedia for growth of the cells. Accordingly, the present inventionprovides cell culture compositions comprising cells of the invention andother components including, but not limited to, media (e.g., a mediadisclosed herein), media components, buffers, chemical compounds,additional cell types, viral material (e.g., viral genomes, viralparticles) and heterologous proteins. In one embodiment, a cell culturecomposition comprises cells of the invention and a media or componentsthereof. Media which may be present in a cell culture compositioninclude serum-free media, serum containing media and APF media. In oneembodiment, a cell composition comprises a media disclosed herein (e.g.,MediV SF101, MediV SF102, MediV SF103, MediV SF104 or MediV SF105) orcomponents thereof.

Methods and Media Formulations

The present invention provides methods and media formulations for thecultivation of non-tumorigenic MDCK cells in serum containing media. Thepresent invention also provides methods for the adaptation to andsubsequent cultivation of non-tumorigenic MDCK cells in serum-free mediaincluding APF media formulations. In certain aspects of the invention,the medias are formulated such that the MDCK cells retain one or more ofthe following characteristics including but limited to, beingnon-tumorigenic, growing as adherent cells, having an epithelial-likemorphology and supporting the replication of various viruses whencultured. It is contemplated that the media formulations disclosedherein or components thereof, may be present in a cell culturecomposition.

Serum containing media formulations are well known in the art. Serumcontaining media formulations include but are not limited to, Dulbecco'sModified Eagle Medium (DMEM)+Fetal Bovine Serum (FBS)+glutamine+glucose.In one embodiment, FBS is present in a serum containing media at aconcentration between about 1% and about 20%, or between about 5% andabout 15%, or between about 5% and about 10%. In a specific embodiment,FBS is present in a serum containing media at a concentration of 10%. Inanother embodiment, glutamine is present in a serum containing media ata concentration of between about 0.5 mM and about 10 mM, or betweenabout 1 mM and 10 mM, or between about 2 mM and 5 mM. In a specificembodiment, glutamine is present in a serum containing media at aconcentration of 4 mM. In still another embodiment, glucose is presentin a serum containing media at a concentration of between about 1 g/Land about 10 g/L, or between about 2 g/L and about 5 g/L. In a specificembodiment, glucose is present in a serum containing media at aconcentration of 4.5 g/L. In yet another embodiment, a serum containingmedia formulation comprises, FBS at a concentration between about 1% andabout 20%, glutamine at a concentration of between about 0.5 mM andabout 10 mM, and glucose a concentration of between about 1 g/L andabout 10 g/L. In a specific embodiment, a serum containing mediaformulation comprises, Dulbecco's Modified Eagle Medium (DMEM)+10% FetalBovine Serum (FBS)+4 mM glutamine+4.5 g/L glucose. DMEM is readilyavailable from numerous commercial sources including, for example,Gibco/BRL (Cat. No. 11965-084). FBS is readily available from numerouscommercial sources including, for example, JRH Biosciences (Cat. No.12107-500M). While FBS is the most commonly applied supplement in animalcell culture media, other serum sources are also routinely used andencompassed by the present invention, including newborn calf, horse andhuman.

In one embodiment, MDCK-S serum adapted non-tumorigenic cells of theinvention are derived from Madin Darby Canine Kidney Cells (MDCK) cellsobtained from the American type Culture Collection (ATCC CCL34) byculturing them in a chemically defined media supplemented with serum. Ina specific embodiment, MDCK cells (ATCC CCL34) are expanded in achemically defined media supplemented with serum to generate the MDCK-Scell line as follows: The MDCK (ATCC CCL34) cells are passaged as needin Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetalbovine serum (10% v/v), 4 mM glutamine and 4.5 g/L glucose to obtainenough cell to prepare a frozen pre Master Cell Bank (PreMCB) designatedMDCK-S. In another specific embodiment, the cells are cultured using theprocess detailed in Example 2, infra. It is specifically contemplatedthat the MDCK-S serum adapted cell are passaged for another 20 passagesor more, from a vial of PreMCB and tested for tumorigenicity in an vivoadult nude mice model and karyology in a karyotype assay. In certainembodiments, the expanded MDCK-S cells will not produce nodules wheninjected subcutaneously into adult nude mice and will have a modalchromosome number of 78 with a range of chromosome numbers of no morethen about 60-88, or of no more then about 65-85, or of no more thanabout 65-80, or of no more then about 70-85. In one embodiment, theMDCK-S cells are non-tumorigenic after at least 20 passages, or after atleast 30 passages, or after at least 40 passages, or after at least 50passages, or after at least 60 passages, or after at least 70 passages,or after at least 80 passages, or after at least 90 passages, or afterat least 100 passages in a medium (e.g., a media described herein).

It will be appreciated by one of skill in the art that the use of serumor animal extracts in tissue culture applications may have drawbacks(Lambert, K. J. et al., In: Animal Cell Biotechnology, Vol 1, Spier, R.E. et al., Eds., Academic Press New York, pp. 85-122 (1985)). Forexample, the chemical composition of these supplements may vary betweenlots, even from a single manufacturer. In addition, supplements ofanimal or human origin may also be contaminated with adventitious agents(e.g., mycoplasma, viruses, and prions). These agents can seriouslyundermine the health of the cultured cells when these contaminatedsupplements are used in cell culture media formulations. Further, theseagents may pose a health risk when substances produced in culturescontaminated with adventitious agents are used in cell therapy and otherclinical applications. A major fear is the presence of prions whichcause spongiform encephalopathies in animals and Creutzfeld-Jakobdisease in humans. Accordingly, the present invention further providesserum-free media formulations.

Serum-free media formulations of the invention include but are notlimited to MediV SF101 (Taub's+Plant Hydrolysate), MediV SF102(Taub's+Lipids), MediV SF103 (Taub's+Lipds+Plant Hydrolysate), MediVSF104 (Taub's+Lipds+Plant Hydrolysate+growth factor) and Medi SF105(same as MediV SF104 except transferrin is replaced with Ferric ammoniumcitrate/Tropolone or Ferric ammonium sulfate/Tropolone). It isspecifically contemplated that Taub's SF medium (Taub and Livingston,1981, Ann NY Acad Sci., 372:406) is a 50:50 mixture of DMEM and Ham'sF12 supplemented with hormones, 5 μg/mL insulin, 5 μg/mL transferrin, 25ng/mL prostaglandin E1, 50 nM hydrocortisone, 5 pM triidothyronine and10 nM Na₂SeO₃, 4.5 g/L glucose, 2.2 g/L NaHCO₃ and 4 mM L-glutamine.

Plant hydrolysates include but are not limited to, hydrolysates from oneor more of the following: corn, cottonseed, pea, soy, malt, potato andwheat. Plant hydrolysates may be produced by enzymatic hydrolysis andgenerally contain a mix of peptides, free amino acids and growthfactors. Plant hydrolysates are readily obtained from a number ofcommercial sources including, for example, Marcor Development, HyCloneand Organo Technie. It is also contemplated that yeast hydrolysates myalso be utilized instead of, or in combination with plant hydrolysates.Yeast hydrolysates are readily obtained from a number of commercialsources including, for example, Sigma-Aldrich, USB Corp, Gibco/BRL andothers.

Lipids that may be used to supplement culture media include but are notlimited to chemically defined animal and plant derived lipid supplementsas well as synthetically derived lipids. Lipids which may be present ina lipid supplement includes but is not limited to, cholesterol,saturated and/or unsaturated fatty acids (e.g., arachidonic, linoleic,linolenic, myristic, oleic, palmitic and stearic acids). Cholesterol maybe present at concentrations between 0.10 mg/ml and 0.40 mg/ml in a 100×stock of lipid supplement. Fatty acids may be present in concentrationsbetween 1 μg/ml and 20 μg/ml in a 100× stock of lipid supplement. Lipidssuitable for media formulations are readily obtained from a number ofcommercial sources including, for example HyClone, Gibco/BRL andSigma-Aldrich.

In one embodiment, Taub's media is supplemented with a plant hydrolysateand a final concentration of at least 0.5 g/L, or at least 1.0 g/L, orat least 1.5 g/L, or at least 2.0 g/L, or at least 2.5 g/L, or at least3.0 g/L, or at least 5.0 g/L, or at least 10 g/L, or at least 20 g/L. Ina specific embodiment, Taub's media is supplemented with a wheathydrolysate. In another specific embodiment, Taub's media issupplemented with a wheat hydrolysate at a final concentration of 2.5g/L. The present invention provides a serum-free media referred toherein as MediV SFM 101 comprising Taub's media supplemented with awheat hydrolysate at a final concentration of 2.5 g/L.

In another embodiment, Taub's media is supplemented with a lipid mixtureat a final concentration of at least 50%, or at least 60%, or at least70%, or at least 80%, or at least 90%, or at least 100%, or at least125%, or at least 150%, or at least 200%, or at least 300% of themanufacturers recommended final concentration. In a specific embodiment,Taub's media is supplemented with a chemically defined lipid mixture. Inanother specific embodiment, Taub's media is supplemented with achemically defined lipid mixture at a final concentration of 100% of themanufacturers recommended final concentration (e.g., a 100× stockobtained from a manufacture would be add to the media to a finalconcentration of 1×). The present invention provides a serum-free mediareferred to herein as MediV SFM 102 comprising Taub's media supplementedwith a chemically defined lipid mixture at a final concentration of 100%of the manufacturers recommended final concentration.

In still another embodiment, Taub's media is supplemented with a planthydrolysate at a final concentration of at least 0.5 g/L, or at least1.0 g/L, or at least 1.5 g/L, or at least 2.0 g/L, or at least 2.5 g/L,or at least 3.0 g/L, or at least 5.0 g/L, or at least 10 g/L, or atleast 20 g/L and with a lipid mixture at a final concentration of atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90%, or at least 100%, or at least 125%, or at least 150%, or atleast 175%, or at least 200% of the manufacturers recommendedconcentration. In a specific embodiment, Taub's media is supplementedwith wheat hydrolysate and a chemically defined lipid mixture. Inanother specific embodiment, Taub's media is supplemented with a wheathydrolysate at a final concentration of 2.5 g/L and a chemically definedlipid mixture at a final concentration of 100% of the manufacturersrecommended final concentration. The present invention provides aserum-free media referred to herein as MediV SFM 103 comprising Taub'smedia supplemented with a wheat hydrolysate at a final concentration of2.5 g/L and a chemically defined lipid mixture at a final concentrationof 100% of the manufacturers recommended final concentration.

In yet another embodiment, Taub's media is supplemented with a growthhormone. Growth hormones which may be used include but are not limitedto, Epidermal Growth Factor (EGF), Insulin Growth Factor (IGF),Transforming Growth Factor (TGF) and Fibroblast Growth Factor (FGF). Ina particular embodiment, the growth hormone is Epidermal Growth Factor(EGF). In one embodiment, Taub's media is supplemented with a growthfactor at a final concentration of between about 0.1 to about 50.0ng/ml, or between about 0.5 to about 25.0 ng/ml, or between about 1.0 toabout 20 ng/ml, or between about 5.0 to about 15.0 ng/ml, or betweenabout 8 ng/ml to about 12 ng/ml. In a specific embodiment, Taub's mediais supplemented with a EGF at a final concentration of about 10 ng/ml.In still other embodiments, Taub's media is supplemented with a growthfactor at a final concentration of between about 0.1 to about 50.0ng/ml, or between about 0.5 to about 25.0 ng/ml, or between about 1.0 toabout 20 ng/ml, or between about 5.0 to about 15.0 ng/ml, or betweenabout 8 ng/ml to about 12 ng/ml and with a plant hydrolysate at a finalconcentration of at least 0.5 g/L, or at least 1.0 g/L, or at least 1.5g/L, or at least 2.0 g/L, or at least 2.5 g/L, or at least 3.0 g/L, orat least 5.0 g/L, or at least 10 g/L, or at least 20 g/L and with alipid mixture at a final concentration of at least 50%, or at least 60%,or at least 70%, or at least 80%, or at least 90%, or at least 100%, orat least 125%, or at least 150%, or at least 175%, or at least 200% ofthe manufacturers recommended concentration. In another specificembodiment, Taub's media is supplemented with a wheat hydrolysate at afinal concentration of 2.5 g/L and a chemically defined lipid mixture ata final concentration of 100% of the manufacturers recommended finalconcentration and EGF at a final concentration of about 10 ng/ml. Thepresent invention provides a serum-free media referred to herein asMediV SFM 104 comprising Taub's media supplemented with a wheathydrolysate at a final concentration of 2.5 g/L and a chemically definedlipid mixture at a final concentration of 100% of the manufacturersrecommended final concentration and EGF at a final concentration ofabout 10 ng/ml.

It will also be appreciated by one skilled in the art that animalprotein-free media formulations may be desirable for the production ofvirus used in the manufacture of vaccines. Accordingly, in certainembodiments one or more or all of the animal derived components of theserum-free media disclosed herein (e.g., MediV SF101, MediV SF102, MediVSF103, MediV SF104 and Medi SF105) is replaced by an animal-freederivative. For example, commercially available recombinant insulinderived from non-animal sources (e.g., Biological Industries Cat. No.01-818-1) may utilized instead of insulin derived from an animal source.Likewise, iron binding agents (see, e.g., U.S. Pat. Nos. 5,045,454;5,118,513; 6,593,140; and PCT publication number WO 01/16294) may beutilized instead of transferrin derived from an animal source. In oneembodiment, serum-free media formulations of the invention comprisetropolone (2-hydroxy-2,4,6-cyclohepatrien-1) and a source of iron (e.g.,ferric ammonium citrate, ferric ammonium sulphate) instead oftransferrin. For example, tropolone or a tropolone derivative will bepresent in an excess molar concentration to the iron present in themedium for at a molar ratio of about 5 to 1 to about 70 to 1, or ofabout 10 to 1 to about 70 to 1. Accordingly, where the ironconcentration in the medium is around 0.3 μM, the tropolone orderivative thereof may be employed at a concentration of about 1.5 μM toabout 20 μM, e.g. about 3 μM to about 20 μM. The iron may be present asferrous or ferric ions, for example resulting from the use of simple orcomplex iron salts in the medium such as ferrous sulphate, ferricchloride, ferric nitrate or in particular ferric ammonium citrate. Thepresent invention provides a serum-free media referred to herein asMediV SFM 105 comprising Taub's media without transferrin supplementedwith a wheat hydrolysate at a final concentration of 2.5 g/L and achemically defined lipid mixture at a final concentration of 100% of themanufacturers recommended final concentration and EGF at a finalconcentration of about 10 ng/ml and Ferric ammonium citrate:Tropolone orFerric ammonium sulfate:Tropolone at a ratio of between 10 to 1 and 70to 1.

In one embodiment, MDCK-SF101, MDCK-SF102, MDCK-SF103, MDCK-SF104 andMDCK-SF105 serum-free adapted non-tumorigenic cells (collectivelyreferred to herein as MDCK-SF) are derived from Madin Darby CanineKidney Cells (MDCK) cells obtained from the American type CultureCollection (ATCC CCL34) by culturing in a chemically defined mediasupplemented with serum for at least one passage and then passaging themin a serum-free media such as, for example, the serum-free mediasdescribed supra. In a specific embodiment, MDCK cells (ATCC CCL34) areadapted to serum-free media to generate a MDCK-SF cell line as follows:The MDCK (ATCC CCL34) cells are passaged in Dulbecco's Modified EagleMedium (DMEM) supplemented with fetal bovine serum (10% v/v), 4 mMglutamine and 4.5 g/L glucose at least once and then passaged inserum-free media. The MDCK-SF cells are then passaged as needed inserum-free media to obtain enough cell to prepare a frozen pre MasterCell Bank (PreMCB). In certain embodiments, the cells are passaged in aserum containing media (e.g., Dulbecco's Modified Eagle Medium (DMEM)supplemented with fetal bovine serum (10% v/v), 4 mM glutamine and 4.5g/L glucose) between 1 and 5 times, or between 4 and 10 time, or between9 and 20 times, or more than 20 times, and then passaged in serum-freemedia (e.g., MediV SF101, MediV SF102, MediV SF103, MediV SF104 and MediSF105).

It is specifically contemplated that the MDCK-SF serum-free adaptedcells are passaged for another 20 passages or more, from a vial ofPreMCB and tested for tumorigenicity in an vivo adult nude mice modeland karyology in a karyotype assay. In certain embodiments, the expandedMDCK-SF cells will not produce nodules when injected subcutaneously intoadult nude mice and/or will have a modal chromosome number of 78. Inanother embodiment, the expanded MDCK-SF cells will have a modalchromosome number of 78 with a range of chromosome numbers of no morethen about 60 to about 88, or of no more then about 65 to about 85, orof no more then about 65-80, or of no more then about 70 to about 85. Inone embodiment, the MDCK-SF cells are non-tumorigenic after at least 20passages, or after at least 30 passages, or after at least 40 passages,or after at least 50 passages, or after at least 60 passages, or afterat least 70 passages, or after at least 80 passages, or after at least90 passages, or after at least 100 passages in a medium (e.g., a mediadescribed herein).

In one embodiment, the serum-free media used for the derivation ofMDCK-SF cells is MediV SF101. In another embodiment, the serum-freemedia used for the derivation of MDCK-SF cells is MediV SF102. In yetanother embodiment, the serum-free media used for the derivation ofMDCK-SF cells is MediV SF103. In still another embodiment, theserum-free media used for the derivation of MDCK-SF cells isMediV-SF104. In another embodiment, the serum-free media used for thederivation of MDCK-SF cells is MediV SF105. In yet another embodiment,the serum-free media used for the derivation of MDCK-SF cells is an APFmedia. It is contemplated that the media described herein may beformulated to eliminate animal proteins. For example bovine transferrinmay be replaced with a recombinant transferrin derived from a non animalsource.

Culture Conditions

The present invention provides methods for the cultivation of MDCK cells(preferably non-tumorigenic) and other animal cells (tumorigenic or not)in serum containing and serum-free media formulations (supra). It isspecifically contemplated that additional culture conditions may play arole in the maintenance of the MDCK-S and MDCK-SF cells in anon-tumorigenic state. These culture conditions include but are notlimited to the choice of adherent surface, cell density, temperature,CO₂ concentration, method of cultivation, dissolved oxygen content andpH.

It is specifically contemplated that one skilled in the art may adaptthe culture conditions in a number of ways to optimize the growth of theMDCK cells of the invention. Such adaptations may also result in aincrease in the production of viral material (e.g., virus),alternatively, one skilled in the art may adapt the culture conditionsto optimize the production of vaccine material from the MDCK cells ofthe invention without regard for the growth of the cells. These cultureconditions include but are not limited to adherent surface, celldensity, temperature, CO₂ concentration, method of cultivation,dissolved oxygen content and pH.

In one embodiment, the MDCK cells of the invention are cultivated asadherent cells on a surface to which they attach. Adherent surfaces onwhich tissue culture cells can be grown on are well known in the art.Adherent surfaces include but are not limited to, surface modifiedpolystyrene plastics, protein coated surfaces (e.g., fibronectin and/orcollagen coated glass/plastic) as well as a large variety ofcommercially available microcarriers (e.g., DEAE-Dextran microcarrierbeads, such as Dormacell, Pfeifer & Langen; Superbead, FlowLaboratories; styrene copolymer-tri-methylamine beads, such as Hillex,SoloHill, Ann Arbor). Microcarrier beads are small spheres (in the rangeof 100-200 microns in diameter) that provide a large surface area foradherent cell growth per volume of cell culture. For example a singleliter of medium can include more than 20 million microcarrier beadsproviding greater than 8000 square centimeters of growth surface. Thechoice of adherent surface is determined by the methods utilized for thecultivation of the MDCK cells of the invention and can be determined byone skilled in the art. Suitable culture vessels which can be employedin the course of the process according to the invention are all vesselsknown to the person skilled in the art, such as, for example, spinnerbottles, roller bottles, fermenters or bioreactors. For commercialproduction of viruses, e.g., for vaccine production, it is oftendesirable to culture the cells in a bioreactor or fermenter. Bioreactorsare available in volumes from under 1 liter to in excess of 100 liters,e.g., Cyto3 Bioreactor (Osmonics, Minnetonka, Minn.); NBS bioreactors(New Brunswick Scientific, Edison, N.J.); laboratory and commercialscale bioreactors from B. Braun Biotech International (B. Braun Biotech,Melsungen, Germany).

In one embodiment, the MDCK cells of the invention are cultivated asadherent cells in a batch culture system. In still another embodiment,the MDCK cells of the invention are cultivated as adherent cells in aperfusion culture system. It is specifically contemplated that the MDCKcells of the invention will be cultured in a perfusion system, (e.g., ina stirred vessel fermenter, using cell retention systems known to theperson skilled in the art, such as, for example, centrifugation,filtration, spin filters and the like) for the production of vaccinematerial (e.g., virus).

In one embodiment, the MDCK cells of the invention are cultivated at aCO₂ concentration of at least 1%, or of at least 2%, or of at least 3%,or of at least 4%, or of at least 5%, or of at least 6%, or of at least7%, or of at least 8%, or of at least 9%, or of at least 10%, or of atleast 20%.

In one embodiment the dissolved oxygen (DO) concentration (pO₂ value) isadvantageously regulated during the cultivation of the MDCK cells of theinvention and is in the range from 5% and 95% (based on the airsaturation), or between 10% and 60%. In a specific embodiment thedissolved oxygen (DO) concentration (pO₂ value) is at least 10%, or atleast 20%, or at least 30%, or at least 50%, or at least 60%.

In another embodiment, the pH of the culture medium used for thecultivation of the MDCK cells of the invention is regulated duringculturing and is in the range from pH 6.4 to pH 8.0, or in the rangefrom pH 6.8 to pH 7.4. In a specific embodiment, the pH of the culturemedium is at least 6.4, or at least 6.6, or at least 6.8, or at least7.0, or at least 7.2, or at least 7.4, or at least 7.6, or at least 7.8,or at least 8.0.

In a further embodiment, the MDCK cells of the invention are cultured ata temperature of 25° C. to 39° C. It is specifically contemplated thatthe culture temperature may be varied depending on the process desired.For example, the MDCK cells of the invention may be grown at 37° C. forproliferation of the cells and at a lower temperature (e.g., 25° C. to35° C.) of for the production of vaccine material (e.g., virus). Inanother embodiment, the cells are cultured at a temperature of less than30° C., or of less than 31° C., or of less than 32° C., or of less than33° C., or of less than 34° C. for the production of vaccine material.

In order to generate vaccine material (e.g., virus) it is specificallycontemplated that the MDCK cells of the invention are cultured such thatthe medium can be readily exchanged (e.g., a perfusion system). Thecells may be cultured to a very high cell density, for example tobetween 1×10⁶ and 25×10⁶ cells/mL. The content of glucose, glutamine,lactate, as well as the pH and pO₂ value in the medium and otherparameters, such as agitation, known to the person skilled in the artcan be readily manipulated during culture of the MDCK cells of theinvention such that the cell density and/or virus production can beoptimized.

Production of Vaccine Material (e.g., Virus)

The present invention provides a process for the production of virusesin cell culture (referred to hereinafter as “the process of theinvention”), in which the MDCK cells of the invention are used. In oneembodiment the process comprises the following steps:

-   -   i) proliferation of the MDCK cells of the present invention in        culture media;    -   ii) infection of the cells with virus; and    -   iii) after a further culturing phase, isolating the viruses        replicated in the non-tumorigenic cells.

In one embodiment the MDCK cells of the invention are proliferated instep (i) as adherent cells. The MDCK cells of the invention can becultured in the course of the process in any media including, but notlimited to, those described supra. In certain embodiments, the MDCKcells of the invention are cultured in the course of the process in aserum-free medium such as, for example, MediV-SF101, MediV-SF102,MediV-SF103, MediV-SF104, MediV-SF105 and APF formulations thereof.Optionally, the MDCK cells of the invention can be cultured in thecourse of the process in a serum containing media (e.g., DMEM+10% FBS+4mM glutamine+4.5 g/L glucose). Additional culture conditions such as,for example, temperature, pH, pO₂, CO₂ concentration, and cell densityare described in detail supra. One skilled in the art can establish acombination of culture conditions for the proliferation of the MDCKcells of the invention for the production of virus.

The temperature for the proliferation of the cells before infection withviruses is in one embodiment between 22° C. and 40° C. In certainembodiments, the temperature for the proliferation of the cells beforeinfection with viruses is less then 39° C., or less than 38° C., or lessthan 37° C., or less than 36° C., or less than 35° C., or less than 34°C., or less than 33° C., or less than 32° C., or less than 30° C., orless than 28° C., or less than 26° C., or less than 24° C. Culturing forproliferation of the cells (step (i)) is carried out in one embodimentof the process in a perfusion system, e.g. in a stirred vesselfermenter, using cell retention systems known to the person skilled inthe art, such as, for example, centrifugation, filtration, spin filters,microcarriers, and the like.

The cells are in this case proliferated for 1 to 20 days, or for 3 to 11days. Exchange of the medium is carried out in the course of this,increasing from 0 to approximately 1 to 5 fermenter volumes per day. Thecells are proliferated up to high cell densities in this manner, forexample up to at least 1×10⁶-25×10⁶ cells/mL. The perfusion rates duringculture in the perfusion system can be regulated via the cell count, thecontent of glucose, glutamine or lactate in the medium and via otherparameters known to the person skilled in the art. Alternatively, thecells in step (i) of the process according to the invention be culturedin a batch process.

In one embodiment of the process according to the invention, the pH, pO₂value, glucose concentration and other parameters of the culture mediumused in step (i) is regulated during culturing as described above usingmethods known to the person skilled in the art.

In another embodiment, the infection of the cells with virus is carriedout at an m.o.i. (multiplicity of infection) of about 0.0001 to about10, or about 0.0005 to about 5, or about 0.002 to about 0.5. In stillanother embodiment, the infection of the cells with virus is carried outat an m.o.i. (multiplicity of infection) of 0.0001 to 10, or 0.0005 to5, or 0.002 to 0.5. After infection, the infected cell culture iscultured further to replicate the viruses, in particular until a maximumcytopathic effect or a maximum amount of virus antigen can be detected.In one embodiment, after infection the cells are cultured at atemperature of between 22° C. and 40° C. In certain embodiments, afterinfection with viruses the cells are cultured at a temperature of lessthen 39° C., or less than 38° C., or less than 37° C., or less than 36°C., or less than 35° C., or less than 34° C., or less than 33° C., orless than 32° C., or less than 30° C., or less than 28° C., or less than26° C., or less than 24° C. In another embodiment, after infection thecells are cultured at a temperature of less than 33° C. In still anotherembodiment, after infection the cells are cultured at a temperature of31° C. In certain embodiments, the culturing of the cells is carried outfor 2 to 10 days. The culturing can be carried out in the perfusionsystem or optionally in the batch process.

The culturing of the cells after infection with viruses (step (iii)) isin turn carried out such that the pH and pO₂ value are maintained asdescribed above. During the culturing of the cells or virus replicationaccording to step (iii) of the process, a substitution of the cellculture medium with freshly prepared medium, medium concentrate or withdefined constituents such as amino acids, vitamins, lipid fractions,phosphates etc. for optimizing the antigen yield is also possible. Thecells can either be slowly diluted by further addition of medium ormedium concentrate over several days or can be incubated during furtherperfusion with medium or medium concentrate. The perfusion rates can inthis case in turn be regulated by means of the cell count, the contentof glucose, glutamine, lactate or lactate dehydrogenase in the medium orother parameters known to the person skilled in the art. A combinationof the perfusion system with a fed-batch process is further possible.

In one embodiment of the process, the harvesting and isolation of theproduced viruses (step (iii)) is carried out after a sufficient periodto produce suitable yields of virus, such as 2 to 10 days, or optionally3 to 7 days, after infection. In one embodiment of the process, theharvesting and isolation of the produced viruses (step (iii)) is carriedout 2 days, or 3 days, or 4 days, or 5 days, or after 6 days, or 7 days,or 8 days, or 9 days, or 10 days, after infection.

Viruses which may be produced in the MDCK cells of the present inventioninclude but are not limited to, animal viruses, including families ofOrthomyxoviridae, Paramyxoviridae, Togaviridae, Herpesviridae,Rhabdoviridae, Retroviridae, Reoviridae, Flaviviridae, Adenoviridae,Picornaviridae, Arenaviridae and Poxyiridae.

Systems for producing influenza viruses in cell culture have also beendeveloped in recent years (See, e.g., Furminger. in Textbook ofInfluenza, ed Nicholson, Webster and Hay, pp. 324-332, Blackwell Science(1998); Merten et al. in Novel Strategies in The Design and Productionof Vaccines, ed Cohen & Shafferman, pp. 141-151, Kluwer Academic(1996)). Typically, these methods involve the infection of suitableimmortalized host cells with a selected strain of virus. Whileeliminating many of the difficulties related to vaccine production inhen's eggs, not all pathogenic strains of influenza grow well and can beproduced according to established tissue culture methods. In addition,many strains with desirable characteristics, e.g., attenuation,temperature sensitivity and cold adaptation, suitable for production oflive attenuated vaccines, have not been successfully grown, especiallyat commercial scale, in tissue culture using established methods.

The present invention provides several non-tumorigenic MDCK cell lines,which have been adapted to grow in either serum containing or serum-freemedias and which are capable of supporting the replication of virusesincluding but not limited to influenza when cultured. These cells linesare suitable for the economical replication of viruses in cell culturefor use as vaccine material. The MDCK cells of the present invention areparticularly useful for the production of cold adapted, temperaturesensitive (ca/ts) strains of influenza (e.g., the influenza strainsfound in FluMist®) which do not grow well using other established celllines (see, Example 1, infra). Further, the MDCK cells of the presentinvention are useful for the production of strains of influenza whichmay not grow in embryonated eggs such as avian influenza viruses whichcan also cause disease in humans (e.g., a “pandemic” strains)

Influenza viruses which may be produced by the process of the inventionin the MDCK cells of the invention include but are not limited to,reassortant viruses that incorporate selected hemagglutinin and/orneuraminidase antigens in the context of an attenuated, temperaturesensitive, cold adapted (ca/ts/att) master strain. For example, virusescan comprise master strains that are one or more of, e.g.,temperature-sensitive (ts), cold-adapted (ca), or an attenuated (att)(e.g., A/Ann Arbor/6/60, B/Ann Arbor/1/66, PR8, B/Leningrad/14/17/55,B/14/5/1, B/USSR/60/69, B/Leningrad/179/86, B/Leningrad/14/55,B/England/2608/76 etc.). Methods for the production of reassortantinfluenza vaccine strains in either eggs or cell lines are known in theart and include, for example, Kilbourne, E. D. in Vaccines (2^(nd)Edition), ed. Plotkin and Mortimer, WB Saunders Co. (1988) and thosedisclosed in PCT Application PCT Patent Publication Nos. WO 05/062820and WO 03/091401. Other influenza viruses which may be produced by theprocess of the invention in the MDCK cells of the invention includerecombinant influenza viruses which may express a heterologous geneproduct, see for example, U.S. Patent Publication Nos. 2004/0241139 and2004/0253273.

In one embodiment, the cells are proliferated (step (i)) as describedsupra, the cells are then infected with influenza viruses (step (ii)).In certain embodiments, the infection is carried out at an m.o.i.(multiplicity of infection) of 0.0001 to 10, or of 0.0005 to 5, or of0.002 to 0.5. In other embodiments, the infection is carried out at anm.o.i. (multiplicity of infection) of about 0.0001 to about 10, or ofabout 0.0005 to about 5, or of about 0.002 to about 0.5. Optionally aprotease is added which brings about the cleavage of the precursorprotein of hemagglutinin [HA₀] and thus the adsorption of the viruses onthe cells. The addition of a protease can be carried out according tothe invention shortly before, simultaneously to or shortly after theinfection of the cells with influenza viruses (step (ii)). If theaddition is carried out simultaneously to the infection, the proteasecan either be added directly to the cell culture to be infected or, forexample, as a concentrate together with the virus inoculate. Theprotease is, in certain aspects of the invention, a serine protease, ora cysteine protease, or an asparagine protease. In one embodiment,trypsin is used. In a specific embodiment, TPCK-treated trypsin is used.

In one embodiment, trypsin is added to the cell culture up to a finalconcentration of 1 to 5000 mU/ml, or 5 to 1000 mU/ml, or 100 to 500mU/ml. In an alternative embodiment, trysin is added to the cell cultureup to a final concentration of 1 to 200 μg/ml, or 5 to 50 μg/ml, or 5 to30 μg/ml in the culture medium. During the further culturing of theinfected cells according to step (iii) of the process according to theinvention, trypsin reactivation can be carried out by fresh addition oftrypsin in the case of the batch process or in the case of the perfusionsystem by continuous addition of a trypsin solution or by intermittentaddition.

After infection, the infected cell culture is cultured further toreplicate the viruses, in particular until a maximum cytopathic effector a maximum amount of virus and/or virus antigen can be detected. Incertain embodiments, the culturing of the cells is carried out for 2 to10 days. The culturing can in turn be carried out in the perfusionsystem or optionally in the batch process. In a further embodiment, thecells are cultured at a temperature of 25° C. to 36° C., or of 29° C. to34° C., after infection with influenza viruses. The culturing of theinfected cells at temperatures below 33° C., in particular in thetemperature ranges indicated above, leads to the production of higheryields of certain influenza viruses, such as, for example B strains.Furthermore, the culturing of the infected cells at temperatures below35° C. is contemplated for the production of temperature sensitive, coldadapted (ts/ca) influenza virus. It is contemplated that ts/ca virusesmay also be attenuated (att). In another embodiment, the cells arecultured at a temperature of less than 30° C., or of less than 31° C.,or of less than 32° C., or of less than 33° C., or of less than 34° C.for the production of ts/ca influenza strains. In a specific embodiment,the cells are cultured at a temperature of 31° C., for the production ofinfluenza virus B strains.

The culturing of the cells after infection with influenza viruses (step(iii)) is in turn carried out, for example, as described supra

In one embodiment of the process, the harvesting and isolation of theproduced influenza viruses (step (iii)) is carried out after asufficient period to produce suitable yields of virus, such as 2 to 10days, or 3 to 7 days, after infection. Viruses are typically recoveredfrom the culture medium, in which infected cells have been grown.Typically crude medium is clarified prior to concentration of influenzaviruses. Common methods include filtration, ultrafiltration, adsorptionon barium sulfate and elution, and centrifugation. For example, crudemedium from infected cultures can first be clarified by centrifugationat, e.g., 1000-2000×g for a time sufficient to remove cell debris andother large particulate matter, e.g., between 10 and 30 minutes.Alternatively, the medium is filtered through a 0.8 μm cellulose acetatefilter to remove intact cells and other large particulate matter.Optionally, the clarified medium supernatant is then centrifuged topellet the influenza viruses, e.g., at 15,000×g, for approximately 3-5hours. Following resuspension of the virus pellet in an appropriatebuffer, such as STE (0.01 M Tris-HCl; 0.15 M NaCl; 0.0001 M EDTA) orphosphate buffered saline (PBS) at pH 7.4, the virus may be concentratedby density gradient centrifugation on sucrose (60%-12%) or potassiumtartrate (50%-10%). Either continuous or step gradients, e.g., a sucrosegradient between 12% and 60% in four 12% steps, are suitable. Thegradients are centrifuged at a speed, and for a time, sufficient for theviruses to concentrate into a visible band for recovery. Alternatively,and for most large scale commercial applications, virus is elutriatedfrom density gradients using a zonal-centrifuge rotor operating incontinuous mode. Additional details sufficient to guide one of skillthrough the preparation of influenza viruses from tissue culture areprovided, e.g., in Furminger, in Textbook of Influenza pp. 324-332Nicholson et al. (ed); Merten et al., in Novel Strategies in Design andProduction of Vaccines pp. 141-151 Cohen & Shafferman (ed), and U.S.Pat. No. 5,690,937. If desired, the recovered viruses can be stored at−80° C. in the presence of a stabilizer, such assucrose-phosphate-glutamate (SPG).

In certain embodiments of the process, the virus is treated withBenzonase® or other a non-specific endonuclease. Optionally, theBenzonase® treatment occurs early in the harvesting and isolation of theproduced influenza viruses (step (iii)). In other embodiments of theprocess, following Benzonase® treatment, the material is clarified.Methods useful for clarification include but are not limited to, directflow filtration (DFF). Additional steps which may be utilized for theharvesting and isolation of the produced influenza virus (step (iii))include but are not limited to, tangential flow filtration (TFF),affinity chromatography as well as ion-exchange chromatography and/orhydroxyapatite chromatography. Other steps are exemplified in theExamples section infra.

Vaccine Compositions and Methods of Use

The invention further relates to viruses (e.g., influenza) which areobtainable by a process of the invention. These viruses can beformulated by known methods to provide a vaccine for administration tohumans or animals. The viruses can be present as intact virus particles(e.g., live attenuated viruses) or as inactive/disintegrated virus(e.g., treated with detergents of formaldehyde). Optionally, a definedviral component (e.g., protein) may be isolated from the viruses bymethods know to the person skilled in the art, and used in thepreparation of a vaccine.

The formulation of intact virus particles (e.g., live attenuatedviruses) may include additional steps including, but not limited to, abuffer exchange by filtration into a final formulation followed by asterilization step. Buffers useful for such a formulation may contain200 mM sucrose and a phosphate or histidine buffer of pH 7.0-7.2 withthe addition of other amino acid excipients such as arginine. In certainembodiments, stabilization protein hydrolysates such as porcine gelatinare added. In some embodiments, the final viral solutions/vaccines ofthe invention can comprise live viruses that are stable in liquid formfor a period of time sufficient to allow storage “in the field” (e.g.,on sale and commercialization when refrigerated at 2-8° C., 4° C., 5°C., etc.) throughout an influenza vaccination season (e.g., typicallyfrom about September through March in the northern hemisphere). Thus,the virus/vaccine compositions are desired to retain their potency or tolose their potency at an acceptable rate over the storage period. Inother embodiments, such solutions/vaccines are stable in liquid form atfrom about 2° C. to about 8° C., e.g., refrigerator temperature. Forexample, methods and compositions for formulating a refrigerator stableattenuated influenza vaccine are described in PCT Patent ApplicationPCT/US2005/035614 filed Oct. 4, 2005, also see PCT Publication WO05/014862. Optionally, spray drying, a rapid drying process whereby theformulation liquid feed is spray atomized into fine droplets under astream of dry heated gas, may be utilized to extend storage time of avaccine formulation. The evaporation of the fine droplets results in drypowders composed of the dissolved solutes (see, e.g., US PatentPublication 2004/0042972). Methods for the generation and formulation ofinactive/disintegrated virus particles for vaccine compositions are wellknown in the art and have been utilized for over 40 years.

Generally, virus or viral components can be administeredprophylactically in an appropriate carrier or excipient to stimulate animmune response specific for one or more strains of virus. Typically,the carrier or excipient is a pharmaceutically acceptable carrier orexcipient, such as sterile water, aqueous saline solution, aqueousbuffered saline solutions, aqueous dextrose solutions, aqueous glycerolsolutions, ethanol or combinations thereof. The preparation of suchsolutions insuring sterility, pH, isotonicity, and stability is effectedaccording to protocols established in the art. Generally, a carrier orexcipient is selected to minimize allergic and other undesirableeffects, and to suit the particular route of administration, e.g.,subcutaneous, intramuscular, intranasal, etc.

Optionally, the formulation for prophylactic administration of theviruses, or components thereof, also contains one or more adjuvants forenhancing the immune response to the influenza antigens. Suitableadjuvants include: saponin, mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil or hydrocarbon emulsions, bacilleCalmette-Guerin (BCG), Corynebacterium parvum, and the syntheticadjuvants QS-21 and MF59.

Generally, vaccine formulations are administered in a quantitysufficient to stimulate an immune response specific for one or morestrains of influenza virus. Preferably, administration of the viruseselicits a protective immune response. Dosages and methods for elicitinga protective immune response against one or more viral strain are knownto those of skill in the art. For example, inactivated influenza virusesare provided in the range of about 1-1000 HID₅₀ (human infectious dose),i.e., about 10⁵-10⁸ pfu (plaque forming units) per dose administered.Alternatively, about 10-50 μg, e.g., about 15 μg HA is administeredwithout an adjuvant, with smaller doses being administered with anadjuvant. Typically, the dose will be adjusted within this range basedon, e.g., age, physical condition, body weight, sex, diet, time ofadministration, and other clinical factors. The prophylactic vaccineformulation is systemically administered, e.g., by subcutaneous orintramuscular injection using a needle and syringe, or a needlelessinjection device. Alternatively, the vaccine formulation is administeredintranasally, either by drops, large particle aerosol (greater thanabout 10 microns), or spray into the upper respiratory tract. While anyof the above routes of delivery results in a protective systemic immuneresponse, intranasal administration confers the added benefit ofeliciting mucosal immunity at the site of entry of the influenza virus.For intranasal administration, attenuated live virus vaccines are oftenpreferred, e.g., an attenuated, cold adapted and/or temperaturesensitive recombinant or reassortant influenza virus. While stimulationof a protective immune response with a single dose is preferred,additional dosages can be administered, by the same or different route,to achieve the desired prophylactic effect. These methods can be adaptedfor any virus including but not limited to, orthomyxoviruses (includinginfluenza A and B strains), paramyxoviruses (including RSV, humanmetapneumovirus and parainfluenza), rhabdoviruses and flavoviruses.

Influenza Virus

The methods, processes and compositions herein primarily concerned withproduction of influenza viruses for vaccines. Influenza viruses are madeup of an internal ribonucleoprotein core containing a segmentedsingle-stranded RNA genome and an outer lipoprotein envelope lined by amatrix protein. Influenza A and influenza B viruses each contain eightsegments of single stranded negative sense RNA. The influenza A genomeencodes eleven polypeptides. Segments 1-3 encode three polypeptides,making up a RNA-dependent RNA polymerase. Segment 1 encodes thepolymerase complex protein PB2. The remaining polymerase proteins PB1and PA are encoded by segment 2 and segment 3, respectively. Inaddition, segment 1 of some influenza strains encodes a small protein,PB1-F2, produced from an alternative reading frame within the PB1 codingregion. Segment 4 encodes the hemagglutinin (HA) surface glycoproteininvolved in cell attachment and entry during infection. Segment 5encodes the nucleocapsid nucleoprotein (NP) polypeptide, the majorstructural component associated with viral RNA. Segment 6 encodes aneuraminidase (NA) envelope glycoprotein. Segment 7 encodes two matrixproteins, designated M1 and M2, which are translated from differentiallyspliced mRNAs. Segment 8 encodes NS1 and NS2, two nonstructuralproteins, which are translated from alternatively spliced mRNA variants.

The eight genome segments of influenza B encode 11 proteins. The threelargest genes code for components of the RNA polymerase, PB1, PB2 andPA. Segment 4 encodes the HA protein. Segment 5 encodes NP. Segment 6encodes the NA protein and the NB protein. Both proteins, NB and NA, aretranslated from overlapping reading frames of a biscistronic mRNA.Segment 7 of influenza B also encodes two proteins: M1 and M2. Thesmallest segment encodes two products, NS1 which is translated from thefull length RNA, and NS2 which is translated from a spliced mRNAvariant.

Reassortant viruses are produced to incorporate selected hemagglutininand neuraminidase antigens in the context of an approved master strainalso called a master donor virus (MDV). FluMist® makes use of approvedcold adapted, attenuated, temperature sensitive MDV strains (e.g.,A/AnnArbor/6/60 and B/Ann Arbor/1/66). A number of methods are usefulfor the generation of reassortant viruses including egg-based methodsand more recently cell culture methods See, e.g., PCT Publications WO03/091401; WO 05/062820 and U.S. Pat. Nos. 6,544,785; 6,649,372;6,951,754). It is contemplated that the MDCK cells, media and processesof the invention are useful for the production of influenza virusesincluding, but not limited to, the influenza strains disclosed herein(e.g., A/AnnArbor/6/60 and B/AnnArbor/1/66) and reassortant virusescomprising genes of the A/AnnArbor/6/60, B/AnnArbor/1/66, PR8. It isfurther contemplated that that the MDCK cells, media and processes ofthe invention are useful for the production of influenza viruses,including reassortant viruses, having one or more of the followingphenotypes, temperature sensitive, cold adapted, attenuated.Reassortants may be generated by classical reassortant techniques, forexample by co-infection methods or optionally by plasmid rescuetechniques (see, e.g., PCT Publications WO 03/091401; WO 05/062820 andU.S. Pat. Nos. 6,544,785; 6,649,372, 6,951,754).

EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples but rather should be construed to encompass any and allvariations which become evident as a result of the teachings providedherein.

Example 1 Determination of Spread of Infection of ca/ts InfluenzaStrains in Cell Lines and Characterization of Influenza Produced in MDCKCells

There has been an effort by the vaccine industry to develop alternativeproduction platforms that do not utilize eggs and to produce influenzavaccines in a mammalian or insect cell culture system. The obviousadvantages are easy scalability, increased process control and removalof egg proteins that could cause allergic reaction in some vaccines.Since cell culture based systems can be rapidly scaled up, it offers anadditional advantage at the time of a influenza pandemic, when there isa potential for shortage of supply of eggs and rapid production ofvaccine is required. Initial studies have been performed with a total of7 different cell lines: 2 human diploid lung fibroblast lines (MRC-5 andWI-38) (data not shown), a human retinoblastoma and a human kidney cellline both of which were genetically constructed for production ofadenoviral products (PER.C6 and 293, respectively) (data not shown), afetal rhesus lung cell line (FRhL2) (data not shown), an African greenmonkey kidney cell line (Vero), and a Marin-Darby canine kidney cellline (MDCK). MDCK cells were the only cell line of those tested to becapable of propagating all four types of cold adapted, temperaturesensitive attenulated (ca/ts/att) reassortant influenza virus strains,H1N1, H3N2, the potential pandemic vaccine strain H5N1, as well as Bstrains, to commercially reasonable titers (>10⁷ Log TCID₅₀/mL) (FIG. 1and data not shown). The genetic and antigenic characteristics of virusgrown in MDCK cells was compared to that of virus grown in eggs. Nosignificant changes in the genomic sequence were seen (data not shown)and the antigenicity as determined by HAI titers were comparable (Table1).

Fluorescent Focus Assay: MDCK and Vero cells were grown in 96 well blackplates over 4 days (DMEM+4 mM glutamine+PEN/Strep). Each well wasinfected with the ca/ts influenza B-strains (B/HongKong/330/01 andB/Yamanashi/166/98) at an MOI of ˜0.01 in DMEM+4 mM glutamine+60 mU/mLTPCK trypsin. The virus infected plates were fixed and immuno-stained asfollows to determine the spread of infection. The medium containingvirus was removed from each plate and the plates washed once with 200μl/well with DPBS (no Ca2+/Mg2+) and the plates were then fixed byaddition of 200 μl/well of cold 4% (v/v) paraformaldehyde in PBS. Theplates were washed twice with 200 μl/well of DPBS (no Ca²⁺/Mg²⁺)followed by incubation of the cells with primary antibody (sheep anti Byamanshi and sheep anti B hongkong diluted in 0.1% saponin, 1% BSA inPBS at a ratio of 1:1000). After incubation for an hour, the primaryantibody was removed and cells were washed thrice with 0.1% Tween 20 inPBS and the wells were incubated with secondary antibody (rabbit antisheep labeled with FITC in 0.1% saponin, 1% BSA in PBS at 1:100 ratiodilution). The wells were visualized daily for 4 days using afluorescence microscope and the images were taken daily using SPOTprogram.

Results And Discussion

A fluorescent focus assay was use to assess whether there was spread ofinfection of ca/ts influenza B-strains in MDCK and Vero and also assessif there was any difference in the spread of virus infection among the50 cell clones of Vero. Since the fluorescence in the monolayerincreased over 4 days in the MDCK cells but not in the Vero cells (see,FIG. 1A), it was concluded that the Vero were not permissive for theproduction of ca/ts B strains while MDCK were. This data was similar tothe data in earlier experiments that showed that B-strains could beproduced to 7-7.5 log₁₀ TCID₅₀ in MDCK cells but only to 4-4.5 log₁₀TCID₅₀ in Vero Cells (data not shown).

The MDCK cells were also tested for their ability to support replicationof a number of ca/ts/att reassortant strains including a potentialpandemic vaccine strain, ca A/Vietnam/1203/2004. MDCK cells wereinfected at a low multiplicity of infection with ca A/Vietnam/1203/2004and virus in the supernatant was quantified at various times postinfection. By 48 hours post infection, the titers of caA/Vietnam/1203/2004 reached approximately 8 log₁₀ TCID₅₀/mL and remainedstable for the next 3 to 4 days. See FIG. 1B and Table 2.

Ca/ts/att strains of type A/H1N1, A/H5N1, A/H3N2 and B replicated torelatively high titers in MDCK cells. In addition, passaging theseca/ts/att strains in MDCK cells did not significantly alter theirgenomic sequence. Three ca/ts/att strains, ca A/Sydney/05/97, caA/Beijing/262/95, and ca B/Ann Arbor/1/94 were passaged once or twice inMDCK cells and the entire coding regions of all 6 internal genes weresequenced and compared to the starting material. No nucleotide changeswere observed (data not shown), demonstrating that this passagingthrough this substrate did not change the genetic composition of thesestrains. Further sequence characterizations is performed on differentvaccine strains produced in MDCK cells under conditions that areexpected to mimic the production process including media composition,input dose (moi), temperature of incubation and time of harvest. Basedon the preliminary data, it is expected that there will be nosignificant changes in the genomic sequence of MDCK-produced virus.

Because the genome was genetically stable following passage in MDCKcell, the biological traits of the vaccine produced in eggs or MDCKcells are expected to be indistinguishable. However, the primary viralproduct from cell culture may have some subtle differences compared tothe egg based product, particularly with respect to post-translationalmodification of viral proteins including HA and NA, or composition oflipids in the viral membrane; both of which could potentially change theoverall physical properties of the virion. Preliminary preclinical dataon the antigenicity of cell culture produced and egg produced vaccinedemonstrated that there were no detectable differences in this importantparameter. Egg stocks of several vaccine strains were passaged throughMDCK cells and the antigenicity of both products was determined bymeasuring the HAI titers using reference antisera. As show in Table 1,all the HAI titers were within 2-fold of one another, indicating thatreplication of the vaccine in cells did not change the antigenicity ofthe vaccine compared to egg derived material.

TABLE 1 HAI Titers of strains produced in eggs and MDCK cells HAI TiterMDCK Strain Egg derived derived A/Panama/20/99 256 256 A/Wuhan/359/951024 2048 A/Wyoming/03/2003 512 1024 B/Jilin/20/2003 64 32 B/HongKong/330/01 64 64 B/Jiangsu/10/2003 128 128

Example 2 Derivation of Non-Tumorigenic Serum MDCK Cells

MDCK cells have been traditionally used for the titration of influenzaviruses (Zambon, 1988, in Textbook of Influenza, ed Nicholson, Websterand Hay, ch 24, pg 324-332, Blackwell Science) and thus could be usedfor the propagation of influenza for the production of vaccinematerials. However, MDCK cells have traditionally been grown in basalmedium formulations like Eagle's Minimal Essential Medium (EMEM)supplemented with FBS. Multiple reports indicate that MDCK cells may betumorigenic when cultivated under these conditions and/or for extendedperiods of time (see for example, Gaush et al., Proc Soc Exp Biol Med,122:931; Leighton et al., 1968, Science, 163:472 and Leighton et al.,1970, Cancer, 26:1022). Thus, there is concern about the use of MDCKcells for the production of vaccine materials and efforts have focusedon the development of other cell lines (e.g., PER.C6 and VERO).Unfortunately, not all influenza strains grow well in other mammaliancell lines, in particular the cold adapted influenza viruses thatcomprise FluMist®, a live attenuated influenza vaccine, only grow toreasonable titers (>10⁷ TCID 50/mL) in MDCK cells (see Example 1,supra). Early reports characterizing MDCK cells indicate that earlypassages of MDCK cells may not be tumorigenic (Gaush et al., 1966, ProcSoc Exp Biol Med. 122:931). It was the goal of this experiment toestablish a culture media and passage protocol to maintain MDCK cells ina non-tumorigenic state.

MDCK cells obtained from the ATCC(CCL 34) were expanded in T-flasksusing DMEM supplemented with 10% FBS, 4 mM glutamine and 4.5 g/L glucoseas the growth medium. A pre-Master MDCK cell bank was established on theserum grown MDCK cells (MDCK-S cells), which was tested forbacterial/fungal contaminants and mycoplasma contamination using routinetests performed by a commercial contractor (BioReliance, Rockville,Md.). The cells were found to be negative for the presence ofbacterial/fungal contaminants. The MDCK-S cells were also found to benegative for the presence of cultivatable mycoplasma. The MDCK-S cellsfrom the bank were also tested by a karyotype assay and found to becanine in origin and had a modal chromosome number of 78 with chromosomenumbers ranging from 70 to 84. The MDCK-S cells were then passaged foranother 20 passages from a vial of PreMCB and tested for karyology andtumorigenicity in an vivo adult nude mice model. The karyology testshowed that late passage MDCK-S cells (p 81/24) showed the same modalchromosome number (78) and range of chromosomes (70 to 84) as the earlypassage MDCK-S cells, showing that the cells did not change on extendedpassaging. 1×10⁷ MDCK-S cells when injected subcutaneously into adultnude mice did not result in the formation of any nodules and were deemedto be non tumorigenic.

Materials and Methods

Materials: MDCK cell (ATCC, Cat. No: CCL-34); T-25, T-75, T-225 flasks(Corning, Cat No.: 430639, 430641, 431082); Dulbecco's Modified Eagle'sMedium (DMEM) powder (Gibco, Grand Island N.Y., Formulation No.:01-5052EF); Fetal Bovine Serum, Gamma-irradiated (JRH, Lenexa Kans.,Cat. No.: 12107-500M); L-Glutamine (JRH, Lenexa Kans., Cat. No.:59202-100M); D-Glucose (Amresco, Cat. No.: 0188-1KG); Dulbecco'sPhosphate buffered saline (DPBS) without Ca²⁺ and Mg²⁺ powder (Gibco,Grand Island N.Y., Cat. No.: 21600-069); 0.05% Trypsin-EDTA (Gibco,Grand Island N.Y., Cat. No.: 25300) Dimethylsulphoxide, DMSO (Sigma, St.Louis Miss., Cat. No.: D2650); 0.4% w/v Trypan blue dye in PBS (Sigma,St. Louis Miss., Cat. No.: T8154); CO₂ Incubator (Form a Scientific,Model No.: 3110); YSI Bioanalyzer (YSI, Model No.: 2700 select); VitroChemistry System (Ortho clinic, Model: DT60 II); Improved Neubaurrhemacytometer (Hausser Scientific, Brightline 0.1 mm deep/Reichert,Brightline 0.1 mm deep).

Subculturing of Serum MDCK (MDCK-S) cells in Tissue Culture Flasks: Avial of serum MDCK cells was obtained from the ATCC. The cells weregrown in DMEM medium supplemented with 10% (v/v) FBS, 4.5 g/L glucose,2.2 g/L NaHCO₃ and 4 mM L-glutamine in T-75 flasks. The cells werepassaged 3 or 4 days postseeding, with a complete medium exchangeperformed on day 3 after seeding if the cells were passaged on day 4.The cells were recovered from T-flasks as described below.

The spent growth medium was removed and the cell monolayer washed twicewith DPBS (calcium and magnesium free). The appropriate amount oftrypsin-EDTA (3 mL/T-75, 7.5 mL/T-225), prewarmed in a 37° C. waterbatch, was added to each flask and the T-flasks incubated in a 37° C.,5% CO₂ incubator for about 15-20 min. The flasks were checked every 5minutes to check if cells had detached and the flasks were rappedseveral times to help detach the cells. When the cells had completeddetached from the T-flask, the trypsin was inhibited by addition ofequal volumes of complete growth medium containing 10% serum (3 mL/T-75,7.5 mL/T-225). The cell suspension was aspirated up and down with anappropriately sized pipette to break any large cell clumps. Two 0.5 mLsamples of cell suspension were counted in a hemacytometer. The cellcounts were repeated if the results of the two counts were not within15% of each other. The cells were diluted to 0.05×10⁶ viable cells/mL infresh warm growth medium (DMEM+10% FBS+4.5 g/L glucose+4 mM glutamine)in fresh flasks and seeded in T-flasks (35 mL/T75 or 100 mL/T-225). Theflasks were then incubated in a 37±1° C., 5% CO₂ environment for 3 daysprior to subculturing or media exchange.

Preparation of MDCK-S cell bank: MDCK-S cells were expanded in T-flasksas described above until the total required amount of cells needed forbanking could be recovered (4×10⁶ cells/vial×number of vials). TheMDCK-S cells were recovered when in the exponential growth phase (3 dayspost seeding) by trypsinization as described. The MDCK-S cellsuspensions from individual flasks were pooled and cells were recoveredby centrifugation at 150-250 g for 7±1 min. The supernatant wasaspirated off from each tube and the cell pellets were resuspended infresh complete growth medium (DMEM+10% FBS+4.5 g/L glucose+4 mMglutamine). The cell suspensions from different centrifuge bottles werepooled and cell suspension was aspirated up and down with a pipetteseveral times to break any large cells clumps. The total cell number wasdetermined and the total number of vials that could be frozen at 4×10⁶cells/vial was determined.

The volume of cell suspension was then adjusted to the above value usingfresh growth medium. Equal volumes of freshly prepared 2× freezingmedium (DMEM+10% FBS+4 mM glutamine+4.5/L glucose+15% DMSO) was added tothe cell suspension. Cell suspension was mixed thoroughly and 1 mL ofcell suspension was dispensed into each cryovial. All the vials weretransferred into Nalgene freezing containers and were placed in a ≦−60°C. freezer. The frozen vials were transferred to a liquid nitrogenstorage tank.

Preparation of MDCK-S cells Growth Curve in T-75 flasks: Cells werepassaged at least 4 times (post thaw) in their growth medium prior tocell growth curve study. MDCK-S cells were expanded into T-225 flasks inorder to obtain at least 2.7×10⁷ total cells. The flasks were grown to80-95% confluent prior to trypsinization as described above. Therecovered MDCK-S cells were pooled and cell suspension aspirated up anddown with a pipette several times to break any large cell clumps. Twosamples (0.5 mL) were removed for cell counts and cell densitydetermined. The two sample counts were repeated if they were not within15% of each other. 2.7×10⁷ total MDCK-S cells were then diluted to atotal volume of 540 mL of complete growth medium (5.0×10⁴ cells/mL).This MDCK-S cell suspension was then dispensed into 14× T-75 flasks (35mL/T-75 flask). The flasks were placed in a 37±1° C., 5% CO₂ incubator.

Two T-flasks were removed daily from incubator for cell counts andmetabolic analysis. Two samples (approximately 1.0 mL) of cell culturemedia were removed from each flask for metabolic analysis. One samplewas used to determine glucose, lactate, glutamine, glutamate and ammoniaconcentrations using the YSI and Vitros analyzers. The other sample wasfrozen at −70° C. for amino acid analysis at a later date. The MDCK-Scells were recovered from each flask by trypsinization as describedabove. The cell density was determined and the total number ofcells/T-flask was also determined. The two counts were repeated if theywere not within 15% of each other. The numbers presented are the averageof two independent growth curves studies performed at two differentpassage numbers (p63 and p65) of MDCK-S cells.

Karyology Test: The karyology test was carried out at Applied GeneticsLaboratories in Melbourne, Fla. Briefly, MDCK-S cells grown in T-225flasks were shipped to Applied Genetics Laboratories. The cells weremaintained and subcultured as per the methods listed above. When thecells were thought to have enough mitotic cells, the cells wereharvested for mitotic analysis. The cells were treated with colcemid(0.02 μg/mL) for 150 minutes at 37° C. The cells were then harvested bytrypsinization, and cells centrifuged for 5 minutes at 200 g. Thesupernatant was aspirated off and the cells resuspended in prewarmedhypotonic solution and incubated at 37° C. for 10 minutes. The swollencells were pelleted by centrifugation and then fixed by incubation incarnoy's solution (3:1 methanol:glacial acetic acid) at room temperaturefor 40 minutes. The cells were again centrifuged and cells washed atleast twice with Carnoy's fixative. After the last centrifugation, thecells were resuspended in 1 to 3 ml of fresh fixative to produce anopalescent cell suspension. Drops of the final cell suspension wereplaced on clean slides and air dried.

Cells were stained by addition of Wright's stain solution in phosphatebuffer to the slides and incubating for 7-10 minutes. The slides werewashed with tap water after 7-10 minutes and then air dried. The cellswere scanned with low power objectives (10×) to find cells in themetaphase stage of cell division and the chromosomes of cells inmetaphase were analyzed via a high power oil immersion lens (100×). A100 cells in metaphase were analyzed for cytogenic abnormalities andchromosome count. 1000 cells were scanned to determine polyploidfrequency and mitotic index (percent of cells under going mitosis).

Sterility Testing of the MDCK-S PRE-MCB (Bacteriostatic and Fungstaticand Four Media Sterility): The MDCK-S Pre-MCB was tested forbacteriostatic and funstatic activity at Bioreliance Inc., Rockville,Md. The assay was performed to meet US 26 and 21 CFR 610.12requirements. This assays tests whether the there is a difference ingrowth of control organisms (Bacillus subtilis, Candida albicans,Clostridium sporogenes, Staphylococcus aureus, Pseudomonas aeruginonsa,Aspergillus Niger) inoculated in appropriate broth medium containing 0.1mL of test sample versus broth medium containing control organisms only.Briefly, the test article was inoculated into three tubes of TSB(soybean-casein digest medium), four tubes of THIO (fluid thioglycollatemedium), two tubes of SAB (Sabourand Dextrose Agar) and one tube of PYG(peptone yeast extract). Each control organism containing less that 100cfu of control organism was then inoculated into the appropriate mediatype. Positive controls consisted of Bacillus subtilis in TSB and THIO,Candida albicans in TSB and SAB (at 20-25° C. and 30-35° C.),Clostridium sporogenes in THIO and PYG, Pseudomonas aeruginosa,Staphyloccus aureus and Aspergillus niger. The negative control wassterile PBS. The media were incubated for 3-5 days and checked forgrowth of organisms.

The test article was also analyzed for presence of bacterial and fungalcontaminants using the four media sterility test at Bioreliance,Rockville Md. and the assay was designed to meet USP 26, EP and21CFR610.12 requirements. Briefly, the test article was inoculated intwo tubes of two tubes of TSB (soybean-casein digest medium), two tubesof THIO (fluid thioglycollate medium), three tubes of SAB (SabourandDextrose Agar) and two tubes of PYG (peptone yeast extract). The mediawere incubated at appropriate temperatures (SAB slants were incubated attwo temperatures) and all tubes observed over a 14 day period with thetubes checked on third/fourth or fifth day, seventh or eight day andfourteenth day of testing. Any test article inoculated tubes whichappeared turbid were plated out and gram stains performed on the plate.Negative controls were sterile PBS.

Mycoplasma/mycoplasmstasis test: A vial of frozen MDCK-S cells (MDCKpreMCB lot no. 747p105) was sent to Bioreliance. The cells were expandedand cultured in T-flasks as explained above. Cell lysates at aconcentration of 5×10⁵ cells/mL were prepared and frozen at −70° C. Thetest article was tested for ability to inhibit growth ofMycoplasmapneumoniae, Mycoplasma orale and Mycoplasma hyorhinis eitherin agar broth/plates and/or in VERO cells.

For the agar isolation assay, the test article was test either spiked orunspiked on agar plates or broth bottles. The test article was spikedwith Mycoplasmapneumoniae and Mycoplasma orale to achieve a dilution of10 to 100 cfu/0.2 mL (for Agar test) and 10 to 100 cfu/10 mL (for semibroth assay). A portion of the test sample was not spiked. 4 semi solidbroth bottles were inoculated with 10 ml each of spiked (2 bottles) orunspiked (2 bottles). One bottle each of spiked/upspiked were incubatedeither aerobically or anaerobically at appropriate temperatures. 10 typeA agar plates and 10 type B agar plates were inoculated with each spikedsample or unspiked sample. Half the type A agar plates and type B agarplates were incubated either aerobically or anaerobically at appropriatetemperatures. Uninoculated mycoplasma semi-solid broth served as theuninoculated negative control. All broth bottles were observed for 21days. Each broth bottle (with exception of uninoculated negativecontrol) was subcultured on days 3, 7 and 14 onto Type A agar plates orType B agar plates (10 plates each, 0.2 mL/plate) and incubated underthe same conditions as the appropriate bottle. They were examined once aday for 21 days.

For the enhanced VERO cell culture assay, the test article was testedspiked or unspiked. The test article was spiked with M. orale and M.hyorhinis at a concentration of 10-100 cfu/0.2 mL. The spiked testarticles, unspiked test articles, positive controls and negativecontrols were each inoculated onto T-75 flasks of VERO cell cultures.After 3-5 days of incubation, the cells from each flask were scraped andsnap frozen. Two tenths of one mL of cell lysate from each flask, wasinoculated into each of well of a six well plate containing VERO cells.In addition positive and negative controls were inoculated intoappropriate wells of six well plates containing VERO cells. After 3-5days the cells were fixed and stained with DNA binding HOECHT dye andevaluated for presence of mycoplasma.

Tumorigenicity test of MDCK-S cells in Nude Mice: Evaluation of tumorformation in nude (nu/nu) athymic mice was performed by BioReliance®,Rockville, Md. Briefly, thirty female athymic mice (4 weeks old) wereinjected subcutaneously with 0.2 mL (1×10⁷ cells/mice) of eitherpositive control (18Cl-10T cells), negative control (Syrian hamsterembryo cells; SHE cells) or the test cells (Serum MDCK cells, 747p105high passage). The animals were randomized before injection and all micewere injected using a 22 gauge needle on the same day. All animals wereobserved every working day and the injection site was palpated twice aweek for lesion development for a period of eighty four days. Eachlesion was measured and the animals were held as long as there was novisible increase in size of the lesion. This was for a maximum of 3months. All mice were sacrificed and necropsied after 84 days and theinjection site, lungs, scapular lymph nodes and gross lesions analyzedby histopathological methods.

Replication of cold adapted influenza strains in MDCK-S: T-75 flaskswere seeded at 5×10⁴ cells/mL (35 mL of DMEM+10% FBS+4 mM glutamine) andgrown in an incubator maintained at 37° C. and 5% CO₂ for 3 days. 3 dayspost seeding, the total cells per T-flask were determined by harvestingusing trypsin EDTA and cell counts determined by Trypan-Blue Exclusion.The remaining T-flasks were then infected as follows. The growth mediawas aspirated off and cells washed twice with 10 mL DPBS (no Ca²⁺/Mg²⁺)per flask. The amount of virus to infect each T-flask at a multiplicityof infection (MOI) of 0.01 was determined as per the equation below:

${{Amount}\mspace{14mu}{of}\mspace{14mu}{virus}\mspace{14mu}({mL})} = \frac{{Total}\mspace{14mu}{Cells}\mspace{14mu}{per}\mspace{14mu}{flask}*M\; O\; I}{10^{\bigwedge}\left( {\log\; T\; C\; I\; D\;{50/{mL}}} \right)}$

MOI being defined as the virus particles per cell added

The required amount of virus is then added to 35 mL of post infectionmedium in each T-flask. (DMEM+4 mM glutamine+60 mu/mL TPCK trypsin). TheT-flasks were then incubated at 33° C., 5% CO₂ and samples taken eachday for 6 days. 10×SP was added to each sample as a stabilizer and thesamples were stored at <−70° C. prior to testing for infectivity.

The concentration of virus present in each sample was determinedaccording to a median tissue culture infectious dose (TCID₅₀) assay thatmeasures infectious virions. Briefly, MDCK cells were grown to confluentmonolayers in 96-well microtiter plates and a serial dilutions of ca/tsinfluenza virus sample was added. The samples in the MDCK cell assayplate were typically at a final dilution of 10⁻⁴ to 10⁻¹⁰. The wells incolumns 1-5 and 8-12 contained virus-diluted sample and wells in columns6-7 received only virus diluent and served as cell controls. This formatproduced two data points (n=2) per plate. Replication of virus in theMDCK cells resulted in cell death and the release of progeny virus intothe culture supernatant. The progeny virus infected other cells,resulting in the eventual destruction of the monolayer. The cytopathiceffect (CPE) resulting from infection was allowed to develop during anincubation at 33±1° C. in a CO₂ environment for a period of six days.The plates were then removed from the incubator, the media in the wellsdiscarded, and 100 μl of MEM+4 mM glutamine+penicillin/streptomycin+MTTwas added to each well. The plates were incubated for 6 hrs at 37° C. 5%CO2 and the number of wells showing CPE was determined by visualinspection of the color formed in each well (yellow/orange signifies CPEwells and solid purple signifying no CPE). The number of wells showingCPE in each half plate was used to calculate the titer (log₁₀ TCID₅₀/mL)based on the Karber modification of the Reed-Muench method.

Results and Discussion

Two frozen vials of serum MDCK cells were thawed in complete growthmedium (DMEM+10% FBS+4 mM glutamine+4.5 g/L glucose) on separateoccasions into T-75 flasks. The cell viability on thaw was 97% and 98%respectively. Cells achieved confluence three days after thawing. Themorphology of cells were epithelial-like and similar to the stockobtained from ATCC (FIG. 3). These cells were passaged 5 times and aPre-master cell bank PreMCB was established for these serum grown MDCKcells (MDCK-S cells). FIG. 2 outlines the process used for thederivation of the MDCK-S pre-master cell bank (pre-MCB).

The growth curves for MDCK-S cells in 10% FBS DMEM medium are showed inFIG. 4. The results are the average of two experiments using cells atdifferent passage numbers (P63&P65). MDCK-S cells had an approximately 1day lag phase where the cell number did not double from seeding(1.75×10⁶ total cell/T75 flask at seeding and 2.9×10⁶ total/T-75 day 1).The glucose consumption/lactate production rate was almost zero for thefirst day showing that the cells were in the lag phase (FIG. 5). Thencells grew exponentially during cell growth period before enteringstationary phase at day 4 post seeding. The doubling time of MCDK-Scells in exponential growth phase was 23.1 hours. During the exponentialphase the glucose consumption and lactate production mirrored each otherwith lactate increasing in concentration as the glucose concentrationdecreased (FIG. 5). The glucose consumption/lactate production ratecorrelated well with the cell growth curve (compare FIGS. 4 and 5). Therates were low during lag phase, increased to 2.93 mM/day for glucose,3.43 mM/day for lactate during the exponential phase from day 1 to day4.

The MDCK-S cells entered into the stationary phase day 4 post seeding,and achieved a maximum cell density was around 29±0.99×10⁶ cell on day 5post seeding (FIG. 4). The cell number remained constant after reachingmaximum density and up to day 7 in this study. The glucose consumptionand lactate production rate slowed to 0.33 mM/day for glucose and 0.25mM/day for lactate in stationary phase. There was still approximately 12mM glucose remaining in the medium after 7 days culture. The ratio ofamount of glucose consumed to lactate produced at day 4 was 1.2.

Glutamine consumption and both glutamate and ammonia production of theMDCK-S cells are shown in FIG. 6. The rate of glutamine consumption andproduction of ammonia correlated with the cell growth curve as well(compare FIGS. 4 and 6). The MDCK-S cells consumed glutamine at a rateof 0.49 mM/day during the exponential growth phase up to day 4 whileproducing ammonia at a rate of 0.32 mM/day up to day 5. Then the rate ofglutamine consumption dropped to 0.24 mM/day while the ammoniaproduction rate dropped to 0.11 mM/day, when the cells entered thestationary phase. The ratio of ammonia production to glutamineconsumption was 0.7 on day 4 post seeding. Glutamate generated fromglutamine metabolism did not accumulate in this 7 days cell growthstudy.

The karyology of the MDCK-S cells was tested at passage 61/4 and passage81/24. The G-band chromosome analysis showed that the cells were caninein origin. The distributions of chromosome number in 100 metaphasescells are shown in FIG. 7. The chromosome count ranged from 70 to 84chromosomes per metaphase for cells at low passage 61/4 and 70 to 84chromosomes for high passage 81/24. Both passages had a modal chromosomenumber of 78 chromosomes. The distribution of chromosomes did not changewith passaging. The modality of cells were as expected for a normalcanine kidney cell (Starke et al., 1972, Prog Immunobiol Stand., 5:178).

The MDCK-S preMCB was tested for presence any bacterial, fungal ormycoplasma contaminants. The pre-MCB was passed sterility test (fourmedia sterility test using direct inoculation method to check bacterialand fungal contaminants) and was found to be negative for presence ofmycoplasma (agar-cultivable and non-agar cultivable assay). The testarticle was also found not to inhibit the growth of positive controls inboth the bacteriostasis/fungistatis test and mycoplasmstatis test.

MDCK-S cells at passage 81/24 (pre-MCB+20 passages) were put on nudemice for tumorigenicity test for 3 months. No neoplasma were diagnosedin any mice that were inoculated with MDCK-S cells demonstrating thatMDCK-S cells were not tumorigenic (Table 4).

The MDCK-S cells were tested and found to be capable of supporting thereplication of cold adapted temperature sensitive attenuated reassortantinfluenza strains (Table 2).

TABLE 2 Growth of cold adapted influenza virus strains in serum andserum-free MediV SF101 adapted MDCK cells Virus Strain Serum MDCKSerum-free MDCK (6:2 reassortant) (log₁₀ TCID₅₀/mL) (log₁₀ TCID₅₀/mL)A/New Caledonia/20/99 8.1 7.8 A/Texas/36/91 6.4 <5.2 A/Panama/2007/996.8 6.4 A/Sydney/05/97 7.0 6.5 B/Brisbane/32/2002 7.2 7.5B/HongKong/330/01 7.2 7.4 B/Victoria/504/2000 6.9 7.5

Example 3 Derivation of Serum-Free MDCK Cells in Taub's Media

The results detailed Example 2 above demonstrate that MDCK cells can becultivated under conditions that maintain their epithelial morphologyand normal karyology as well as their ability to replicate cold adaptedinfluenza strains. In addition, we demonstrated that cultivation of MDCKcells under the conditions developed in the above study results in MDCKcells that are non-tumorigenic. However, the culture medium used inExample 2 contains fetal bovine serum (FBS). FBS is a complex mixture ofconstituents and there have been problems reported of lot-to-lotvariation. Also, the ongoing problems with bovine spongiformencephalopathy (BSE) in cows raise safety concerns. The development ofserum-free medium in which the non-tumorigenic nature and growthcharacteristics of the MDCK-S cell line is maintained is important forincreasing the safety of biologicals produced for therapy andvaccination.

Madin Darby Canine Kidney Cells (MDCK) cells obtained from the ATCC (ccl34) were expanded in T-flasks using DMEM supplemented with 10% FBS, 4 mMglutamine and 4.5 g/L glucose as the growth medium for 5 passages. Thecells were then transferred to serum-free Taub's media (see below forformulation). The cells adapted to grow in the Taub's media formulationswere designated MDCK-T. A pre-MCB was established for the MDCK-T cells(see FIG. 8) and was tested for bacterial/fungal contaminants andmycoplasma contamination. The cells the MDCK-T cell pre-Master cell bankwere also tested by a karyotype assay found to be canine in origin andhad a modal chromosome number of 78 with chromosome numbers ranging from52 to 84. In addition, the MDCK-T cells were passaged for at leastanother 20 passages from a vial of PreMCB and tested for karyology andtumorigenicity in an vivo adult nude mice model. However, the MDCK-Tcells were found to be tumorigenic in this model indicating that thepublished Taub's media did not support the stable cultivation of MDCKcells for the production of human vaccine material.

Materials and Methods

Materials: MDCK cell (ATCC, Cat. No: CCL-34, passage 54); T-25, T-75,T-225 flasks (Corning, Cat No.: 430639, 430641, 431082); Dulbecco'sModified Eagle's Medium (DMEM) powder (Gibco, Grand Island N.Y.,Formulation No.: 01-5052EF); Ham F12 Nutrients mixture powder (Gibco,Grand Island N.Y., Cat. No.: 21700-075); Fetal Bovine Serum,Gamma-irradiated (JRH, Lenexa Kans., Cat. No.: 12107-500M); L-Glutamine(JRH, Lenexa Kans., Cat. No.: 59202-100M); D-Glucose (Amresco, Cat. No.:0188-1KG); Dulbecco's Phosphate buffered saline (DPBS) without Ca²⁺ andMg²⁺ powder (Gibco, Grand Island N.Y., Cat. No.: 21600-069); Insulinpowder (Serological, Cat. No. 4506); Transferrin (APO form) (Gibco,Grand Island N.Y., Cat. No.: 11108-016); Prostaglandin E1 (Sigma, St.Louis Miss., Cat. No.: P7527); Hydrocortisone (Mallinckrodt, Cat. No.:8830(-05)); Triidothyronine (Sigma, St. Louis Miss., Cat. No.: T5516);Sodium Selenium (EMD, Cat. No.: 6607-31); 0.05% Trypsin-EDTA (Gibco,Grand Island N.Y., Cat. No.: 25300); Lima bean trypsin inhibitor(Worthington, Cat. No.: LS002829); Dimethylsulphoxide, DMSO (Sigma, St.Louis Miss., Cat. No.: D2650); 0.4% w/v Trypan blue dye in PBS (Sigma,St. Louis Miss., Cat. No.: T8154); Improved Neubaurr hemacytometer(Hausser Scientific, Brightline 0.1 mm deep/Reichert, Brightline 0.1 mmdeep); YSI Bioanalyzer (YSI, Model No.: 2700 select); Vitro ChemistrySystem (Ortho clinic, Model: DT60 II).

Formulation of Taub's Serum free Media: Taub's media (Taub andLivingston, 1981, Ann NY Acad Sci., 372:406) is a serum-free mediaformulation that consists of DMEM/HAM F12 (1:1) containing 4.5 g/Lglucose and 4 mM glutamine as the basal media formulation, to which thehormones/factors are added as indicated in Table 3.

TABLE 3 Hormones and growth factors added to serum-free mediaformulations Name of Component Final Concentration Insulin 5 μg/mLTransferrin 5 μg/mL Triiodothyronine (T₃) 5 × 10⁻¹² M  Hydrocortisone 5× 10⁻⁸ M Prostaglandin E₁ 25 ng/mL  Sodium Selenite    10⁻⁸ M

Taub's SFM is made fresh at the time of passaging or refeed by theaddition of stock solutions of hormone supplements to SF DMEM/Ham F12medium+4 mM glutamine+4.5 g/L glucose+10⁻⁸ M sodium selenite. 100 mL ofTaubs Media is made by addition of 100 μL of insulin stock (5 mg/mL)solution, 100 μL transferrin stock solution (5 mg/mL), 100 uLtriiodothyronine (T3) stock solution (5×10⁻⁹ M), 5 μL of hydrocortisonestock solution (10⁻³ M) and 50 μL of prostaglandin E1 stock solution (50μg/mL) to basal DMEM/Ham F12 medium+4 mM glutamine+4.5 g/L glucose+10⁻⁸M sodium selenite. All stocks solutions are prepared as follows:

Insulin Stock Solution—A 5 mg/ml stock solution is made by dissolvingthe appropriate amount of insulin in 0.01 N HCl. The solution is passedthrough a 0.2 micron sterilizing grade filter and aliquoted into Nalgenecryovial and stored at 4° C.

Transferrin Stock Solution—A 5 mg/ml stock solution is made bydissolving the appropriate amount of transferrin in MilliQ water. Thesolution is passed through a sterilizing grade filter and then aliquotedinto Nalgene cryovial and store <−20° C.

Triiodothyronine (T₃) Stock Solution—A stock solution is made bydissolving the appropriate amount of T3 in 0.02 N NaOH to obtain a 10⁻⁴M solution. This is stock solution is further diluted to a concentrationof 5×10⁻⁹ M stock solution with 0.02 N NaOH, passed through asterilizing grade filter, aliquoted into Nalgene cryovial and stored at<−20° C.

Hydrocortisone Stock Solution—A 10⁻³ M stock solution is made bydissolving the appropriate amount of hydrocortisone in 100% EtOH andaliquoted into Nalgene cryovials. The vials are stored at 4° C. for 3-4months.

Prostaglandin E₁ Stock Solution—A 50 μg/mL stock solution made bydissolving the appropriate amount of PGE1 in 100% sterile EtOH andaliquoted into Nalgene cryovial and stored at <−20° C.

Na₂SeO₃ Stock Solution—A 10⁻² M stock solution is made by dissolving theappropriate amount of sodium selenide in WFI water or MilliQ water. Thisis further diluted in water to a final concentration of 10⁻⁵ M passedthrough a sterilizing grade filter and stored at 4° C.

Adaptation of MDCK-S cells into Serum free Taub's media: A frozen vialof MDCK cells from ATCC (passage 54) was grown in 10% FBS DMEM mediumwith 4.5 g/L glucose, 2.2 g/L NaHCO₃ and 4 mM L-glutamine for 5 passages(as described above) before passaging into a serum-free Taub's media.Serum MDCK grown in a T-75 flask were recovered by trypsinization. Thespent growth medium was removed and cell monolayer washed twice withDPBS (calcium and magnesium free) and then DPBS was discarded. Theappropriate amount of pre-warmed trypsin-EDTA (3 mL/T-75) was added andthe T-flask was incubated in a 37° C., 5% CO₂ incubator for about 15min. The flasks were rapped against the palm of the hand several timesto completely detach the cells. Equal volume of lima bean trypsininhibitor was added to neutralize the trypsin and two samples were takento determine concentration of cells in the cell suspension. 1.75×10⁶cells were then diluted into 35 mL Taub's media in a fresh T75 flask.The flask was placed in an cell culture incubator maintained at 5% CO₂,37±1° C. The cells were either subcultured 3 days post seeding or acomplete medium exchange was performed on day 3 followed by subculturingon day 4 postseeding.

Subculturing of Taub's media Adapted MDCK cells: The spent growth mediumwas removed and cell monolayer washed twice with DPBS (calcium andmagnesium free). The appropriate amount of pre-warmed trypsin-EDTA (3mL/T-75, 7.5 mL/T-225) was added and the T-flask was incubated in a 37°C., 5% CO₂ incubator for about 15 min. The flasks were rapped againstthe palm of the hand several times to completely detach the cells. Thetrypsin was then inhibited by addition of equal volumes of lima beantrypsin inhibitor (3 mL/T-75, 7.5 mL/T-225). The cell suspension washomogenized by aspirating up and down with an appropriately sizedpipette. Two 0.5 mL samples of cell suspension were taken for cellcounting. The cell counts were repeated if the results of the two countswere not within 15% of each other. After counting, the cells werediluted to 0.05×10⁶ viable cells/mL in fresh prewarmed Taub's media infresh flasks, for a total volume of 35 mL/T75 or 100 mL/T-225. Theflasks were then incubated in a 37±1° C., 5% CO₂ environment. Cells wereeither subcultured to new T-flasks on day 3 (as described below) or acomplete media exchange was performed and the culture subcultured to newT-flasks on day 4 post seeding.

Preparation of Taub's media Adapted MDCK cell PreMCB Banks: Thepre-master cell banks for the Taub's serum-free adapted MDCK cell line(MDCK-T) were prepared as described in Example 2 above, except that the2× freezing medium was Taub's media+15% DMSO.

Characterization of Taub's media Adapted MDCK (MDCK-T) cells: Karyology,sterility and mycoplasma testing of the MDCK-T preMCB was performed asdescribed in Example 2 except that Taub's media was used in place ofserum containing complete media. In addition the growth curvecharacteristics of MDCK-T cells in T-75 flasks and the replication ofcold adapted influenza strains in MDCK-T cells were examined asdescribed in Example 2 except that Taub's media was used in place ofserum containing complete media. Tumorigenicity studies were performedon MDCK-T cells at passage 88/29 (pre-MCB+20 passages) by BioReliance asdescribed in Example 2 above.

Results and Discussion

A frozen vial of MDCK-T preMCB (passage 64/5) cells was thawed intoserum-free Taub's media in T-75 flasks. The cell viability was 97% and5.25×10⁶ cells were recovered from frozen vial upon thawing. Cells wereconfluent three days after thawing. Cell morphology showedepithelia-like cells similar to the parent MDCK-S cells. (FIG. 9).

The growth curves for MDCK-T cells in Taub's SF medium are showed inFIG. 10. The results are the average of two experiments using cells atdifferent passage numbers (P71/12 & P73/14). MDCK-T cells had no lagphase with cells doubling one day post seeding (3.42×10⁶ total cell/T75flask day 1 versus 1.75×10⁶ total cell/T75 flask on day 0). The cellswere in the exponential phase of growth till day 4, when they enteredinto the stationary phase. The doubling time of cells in the exponentialphase was 20.4 hrs. During the exponential phase (day 0 to day 4) theyutilized glucose and glutamine (FIGS. 11 and 12) while producing lactateand ammonia. The glucose consumption/lactate production rate correlatedwell with the cell growth curve (compare FIGS. 10 and 11). The glucoseconsumption rate was 1.78 mM/day during the exponential phase from day 0to day 4 and lactate was produced at a rate of 2.88 mM/day. MDCK-T cellsonly consumed about a total of 10 mM glucose in the medium up to 7 daysculture. The ratio of amount of glucose consumed to lactate produced atday 4 post seeding was 1.2. The rate of glucose consumption and lactateproduction slowed down after day 4 when cells entered into thestationary phase, with the glucose consumption being 0.65 mM/day andlactate being produced at a rate of 0.46 mM/day. The maximum celldensity of 37±0.24×10⁶ was achieved around day 4 post seeding. The celldensity did not drop during the stationary phase and remained constanttill day 7.

The glutamine consumption rate and ammonia production rate were similarto the MDCK-T cell growth and glucose/lactate profiles (compare FIGS.10, 11 and 12). The MDCK-T cells consumed glutamine at a rate of 0.36mM/day during the exponential growth phase (day 0 to day 4) with therate dropping to 0.27 mM/day when the cells entered the stationary phase(day 4 to day 7). Ammonia production increased linearly up to day 7 atrate of 0.22 mM/day. The ratio of ammonia production to glutamineconsumption was 0.49 on day 4 post seeding. Glutamate concentration didnot change appreciably during the entire 7 day period.

MDCK-T cells were tested for their ability to support ca/ts influenzareplication as per example 2. The results shown in Table 2 indicate thatMDCK-T cells were able to support the replication of ca/ts influenzareplications to levels nearly the same as seen for the MDCK-S cells.

MDCK-T cell karyology was tested at passage 68/9 and passage 88/29. TheG-band chromosome analysis showed that the cells were canine in origin.The distributions of chromosome number in 100 metaphases cells wereshown in FIG. 13. The chromosome count ranged from 52 to 82 chromosomesper metaphase for cells at low passage 68/9, range from 54 to 82chromosomes for high passage 81/24 indicating that the distribution ofchromosomes did not change with passaging. However, it can be seen thatthe MDCK-T cells show a wider spread in chromosome number (52 to 84) ascompared to the MDCK-S cells (70-84).

The MDCK-T preMCB was tested for presence any bacterial, fungal ormycoplasma contaminants. The MDCK-T pre-MCB was passed sterility test(four media sterility test using direct inoculation method to checkbacterial and fungal contaminants) and was found to be negative forpresence of mycoplasma (agar-cultivable and non-agar cultivable assay).The test article was also found not to inhibit the growth of positivecontrols in both the bacteriostasis/fungistatis test and mycoplasmstatistest.

MDCK-T cells at passage 88/29 (pre-MCB+20 passages) were put on nudemice for tumorigenicity test for 3 months. The test article wasdiagnosed as adenocarcinomas at the site of injection in six of ten testarticle mice. This shows that the MDCK cells grown in SF Taubs media aretumorigenic. The tumorigenicity, estimated TP50 and karyology for MDCK-Sand MDCK-T cells is summarized in Table 4 below.

Example 4 Derivation of Serum-Free MDCK Cells in MediV Serum-Free Medias

The results detailed in Example 3 demonstrate that, although MDCK cellsadapted to grow in serum-free Taub's media (MDCK-T) had excellent growthcharacteristics and were able to support the replication of ca/tsinfluenza strains, they were tumorigenic. Thus, these results indicatethat MDCK cells can readily become transformed in the standardserum-free media formulations reported in the literature. In accordancewith the invention, several additional serum-free media formulationswere developed and tested for their ability to maintain thenon-tumorigenic nature of the MDCK-S cells. MDCK-S cells were adapted toeach of the new serum-free formulations designated MediV SFM 101, 102and 103. These serum-free adapted cell lines were designated MDCK-SF101,-SF102 and -SF103, respectively and are referred to as “MDCK-SF”,collectively. PreMCBs were generated for each MDCK-SF adapted cell line.The MDCK-SF cell line preMCBs were tested for bacterial/fungalcontaminants and mycoplasma contamination (awaiting final results). TheMDCK-SF preMCBs were also tested by a karyotype assay, MDCK-SF101 andMDCK-SF102 cells had a modal chromosome number of 78 with chromosomenumbers ranging from and 70 to 82 and 60 to 80, respectively. Inaddition, the cells from each serum-free media bank were passaged for atleast another 20 passages from a vial of PreMCB and MDCK-SF103 wastested for karyology and tumorigenicity in an vivo adult nude micemodel. At passage 87 MDCK-SF103 was found to have a modal chromosomenumber of 78 ranging from 66 to 80 and were deemed to be nontumorigenic.

Materials: MDCK cell (ATCC, Cat. No: CCL-34, passage 54); T-25, T-75,T-225 flasks (Corning, Cat No.: 430639, 430641, 431082); Dulbecco'sModified Eagle's Medium (DMEM) powder (Gibco, Grand Island N.Y.,Formulation No.: 01-5052EF); Ham F12 Nutrients mixture powder (Gibco,Grand Island N.Y., Cat. No.: 21700-075); Fetal Bovine Serum,Gamma-irradiated (JRH, Lenexa Kans., Cat. No.: 12107-500M); L-Glutamine(JRH, Lenexa Kans., Cat. No.: 59202-100M); D-Glucose (Amresco, Cat. No.:0188-1KG); Dulbecco's Phosphate buffered saline (DPBS) without Ca²⁺ andMg²⁺ powder (Gibco, Grand Island N.Y., Cat. No.: 21600-069); Insulinpowder (Serological, Cat. No. 4506); Transferrin (APO form) (Gibco,Grand Island N.Y., Cat. No.: 11108-016); Prostaglandin E1 (Sigma, St.Louis Miss., Cat. No.: P7527); Hydrocortisone (Mallinckrodt, Cat. No.:8830(-05)); Triidothyronine (Sigma, St. Louis Miss., Cat. No.: T5516);Sodium Selenium (EMD, Cat. No.: 6607-31); 0.05% Trypsin-EDTA (Gibco,Grand Island N.Y., Cat. No.: 25300); Lima bean trypsin inhibitor(Worthington, Cat. No.: LS002829); Dimethylsulphoxide, DMSO (Sigma, St.Louis Miss., Cat. No.: D2650); 0.4% w/v Trypan blue dye in PBS (Sigma,St. Louis Miss., Cat. No.: T8154); Improved Neubaurr hemacytometer(Hausser Scientific, Brightline 0.1 mm deep/Reichert, Brightline 0.1 mmdeep); YSI Bioanalyzer (YSI, Model No.: 2700 select); Vitro ChemistrySystem (Ortho clinic, Model: DT60 II).

Formulation of MediV Serum free Medias (MediV SFM 101, 102 and 103):Each MediV serum-free media formulation uses Taub's media (see themethods section of example 2 above) as a basal media and addssupplements as follows:

-   -   MediV SFM 101: Taub's+2.5 g/L Wheat Peptone E1 from Organo        Techine (cat no 19559). Wheat Peptone E1 is stored in water as a        sterile 250 g/L stock solution.    -   MediV SFM 102: Taub's+100× chemically defined lipid concentrate        from GIBCO BRL (cat no. 11905) added to a final concentration of        1×.    -   MediV SFM 103: Taub's+1× final concentration lipid concentrate        from GIBCO+2.5 g/L Wheat Peptone E1 from Organo Technie.    -   Medi SFM 104: Taub's+1× final concentration lipid concentrate        from GIBCO+2.5 g/L Wheat Peptone E1 from Organo Technie+0.01        μg/mL EGF (multiple sources).    -   Medi SFM105: Taub's without Transferrin, +1× final concentration        lipid concentrate from GIBCO+2.5 g/L Wheat Peptone E1 from        Organo Technie+0.01 ug/mL EGF+Ferric ammonium citrate:Tropolone        or Ferric ammonium sulfate:Tropolone at a ratio of between 10 to        1 and 70 to 1.

Adaptation of MDCK-S cells into Serum free MediV SFM media formulations:A frozen vial of MDCK cell from ATCC was grown in 10% FBS DMEM mediumwith 4.5 g/L glucose, 2.2 g/L NaHCO₃ and 4 mM L-glutamine for 5 passages(as described above) before passaging into a MediV SFM media formulation(MediV SFM 101, MediV SFM 102 or MediV SFM 103). Serum MDCK grown in aT-75 flask were recovered by trypsinization. The spent growth medium wasremoved and cell monolayer washed twice with DPBS (calcium and magnesiumfree) and then DPBS was discarded. The appropriate amount of pre-warmedtrypsin-EDTA (3 mL/T-75) was added and the T-flask was incubated in a37° C., 5% CO₂ incubator for about 15 min. The flasks were rappedagainst the palm of the hand several times to completely detach thecells. Equal volume of lima bean trypsin inhibitor was added toneutralize the trypsin and two samples were taken to determineconcentration of cells in the cell suspension. 1.75×10⁶ cells were thendiluted into 35 mL of the desired MediV SFM media formulation in a freshT75 flask. The flask was placed in an cell culture incubator maintainedat 5% CO₂, 37±1° C. The cells were either subcultured 3 days postseeding or a complete medium exchange was performed on day 3 followed bysubculturing on day 4 postseeding. Cells maybe adapted to MediV SF104and MediV SF105 using the same procedure described above.

Subculturing of MediV SFM media Adapted MDCK cells: The spent growthmedium was removed and cell monolayer washed twice with DPBS (calciumand magnesium free). The appropriate amount of pre-warmed trypsin-EDTA(3 mL/T-75, 7.5 mL/T-225) was added and the T-flask was incubated in a37° C., 5% CO₂ incubator for about 15 min. The flasks were rappedagainst the palm of the hand several times to completely detach thecells. The trypsin was then inhibited by addition of equal volumes oflima bean trypsin inhibitor (3 mL/T-75, 7.5 mL/T-225). The cellsuspension was homogenized by aspirating up and down with anappropriately sized pipette. Two 0.5 mL samples of cell suspension weretaken for cell counting. The cell counts were repeated if the results ofthe two counts were not within 15% of each other. After counting, thecells were diluted to 0.05×10⁶ viable cells/mL in the appropriate freshprewarmed MediV SFM media formulation in fresh flasks, for a totalvolume of 35 mL/T75 or 100 mL/T-225. The flasks were then incubated in a37±1° C., 5% CO₂ environment. Cells were either subcultured to newT-flasks on day 3 (as described below) or a complete media exchange wasperformed and the culture subcultured to new T-flasks on day 4 postseeding. Note: MDCK-SF cells are always subcultured into the same MediVSFM media formulation as they were adapted to.

Preparation of MediV SFM media Adapted MDCK cell PreMCB Banks: Thepre-master cell banks for the serum-free adapted MDCK cell lines areprepared as described in example 1 above, except that the 2× freezingmedium is the appropriate MediV SFM media formulation+15% DMSO.

Characterization of MediV SFM media Adapted MDCK (MDCK-SF) cells:Karyology, sterility and mycoplasma testing of the MDCK-SF preMCBs aretested according to methodology described herein, e.g., in Example 2except that the appropriate MediV SFM media formulation is used in placeof serum containing complete media. Further, the growth curvecharacteristics of MDCK-SF cells in T-75 flasks and the replication ofcold adapted influenza strains in MDCK-SF cells can be examined asdescribed in Example 2 except that the appropriate MediV SFM mediaformulation is used in place of serum containing complete media. Inaddition, tumorigenicity studies can be performed on MDCK-SF cells afteran additional number of passages (e.g., preMCB+20 passages) by acommercial contractor (e.g., BioReliance) as described in Example 2above.

Results and Discussion

The cell karyology of MDCK-SF101 and MDCK-SF102 cells was tested atpassage 71/9 and of MDCK-SF103 at passage 87. The distributions ofchromosome number in 100 metaphases of MDCK-T, MDCK-SF101 and MDCK-SF102cells are shown in FIG. 14 and of MDCK-SF103 in FIG. 19. It can be seenthat the MDCK-T cells show a wider spread in chromosome number (52 to84) as compared to MDCK-SF101, MDCK-SF102 or MDCK-SF103 cells (70-82,60-80, and 66-80 respectively). The spread in chromosome number for theMDCK-SF101, MDCK-SF102 and MDCK-SF103 cells is much closer to that seenfor the non-tumorigenic MDCK-S serum grown cells (70-84) indicating thatthe MediV SF101, MediV SF102, and MediV SF103 media formulations arebetter able to maintain the normal chromosomal number of MDCK cellsgrown in these formulations.

A representative preliminary growth curve for MDCK-SF103 cells in MediVSF103 medium is showed in FIG. 16. MDCK-SF103 cells had about a one daylag phase. The cells were in the exponential phase of growth until aboutday 4, when they entered into the stationary phase. During theexponential phase (day 0 to day 4) they utilized glucose and glutamine(FIGS. 17 and 18) while producing lactate and ammonia. The glucoseconsumption/lactate production rate correlated well with the cell growthcurve (see FIGS. 16 and 17). The maximum cell density of ˜17×10⁶ wasachieved around day 4 post seeding. The cell density did not drop duringthe stationary phase and remained fairly constant till day 7.

The glutamine consumption rate and ammonia production rate were similarto the MDCK-SF103 cell growth and glucose/lactate profiles (see FIG.18). Ammonia production increased linearly up to day 7 while theglutamate concentration did not change appreciably during the 7 dayperiod.

MDCK-SF103 cells were tested for their ability to support thereplication of several reassortant influenza strains as described inExample 7 below. The results shown in FIG. 20A indicate that MDCK-SF103cells were able to support the replication of each influenza straintested.

The MDCK-SF103 cells were put on nude mice for tumorigenicity test for 3months as described above. The test article was deemed to benon-tumorigenic in the adult nude mouse model RioReliance Study NumberAB09EU.001000.BSV).

TABLE 4 Tumorigenicity and Karyology of MDCK cells passed in differentmedias. Estimated TP₅₀* Cells (no animals with (passage tumors/totalKaryology number) Tumorigenicity animals) Median number; comments MDCK-SND ND 78; Few cells with anomalous (P61/4) chromosome number (70 to 82)MDCK-S No neoplasias. Not estimable 78; Few cells with anomalous(P81/24) Fibrosarcomas (>10⁷) chromosome number (70 to 82) at injectionsite (0/10) MDCK-T ND ND 78; Large distribution of cells (P63/4) withchromosome number of 52 to 82 MDCK-T Neoplasias ~10⁷ 78; Largedistribution of cells (P88/29)) noted (6/10) with chromosome number of52-82 MDCK-SF101 ND ND 78; Few cells with anomalous chromosome number(70 to 82) MDCK-SF102 ND ND 78; Few cells with anomalous chromosomenumber (60 to 80) MDCK-SF103 No neoplasias. Not estimable 78; Few cellswith anomalous Fibrosarcomas (>10⁷) chromosome number (66 to 80) atinjection site (0/10) *TP₅₀: Number of cells required to induce tumorsin 50% of animals ND: Not done

Example 5 Infection of Human Epithelial Cells in Culture

To evaluate the biochemical, biological, and structural similaritiesfollowing replication of the MDCK and egg produced vaccines in cells ofhuman origin, vaccines is passaged once in relevant diploid human cells,such as normal human bronchial epithelial cells (NHBE). This passageserves to mimic a single infection event in the human airway and thenenable comparison of the progeny virus, the virus that is ultimatelyresponsible for eliciting an effective immune response. Studies of thevaccines' hemagglutinin (binding and fusion) and neuraminidaseactivities are measured on these materials as well as other biochemicaland structural studies including electron microscopy, infectious tototal particle ratios, and viral genome equivalents are evaluated.Overall, these comparisons serve to demonstrate the comparability of thecell-derived vaccine to the effective and safe egg produced vaccine.Methods for testing for the presence of bacterial and fungalcontaminants are well known in the art and routinely performed bycommercial contractors (e.g., BioReliance®, Rockville, Md.). A summaryof analytical studies which may be performed is summarized in Table 5.

TABLE 5 Preclinical Studies To Compare Cell And Egg Produced Vaccines Invivo (ferrets) In vitro* Attenuation/Replication Virus binding Extent ofreplication in upper airway Hemagglutination titer Kinetics ofreplication in upper airway Binding of different sialic acidsImmunogenicity Phyical properties Cross-reactivity Morphology by EMKinetics Infectious: Total particles (genomes) Infectivity Fusionactivity Dose required for detectable replication pH optimum Doserequired for antibody response temperature optimum Genomic sequenceNeuraminidase activity

Example 6 Production, Testing and Characterization of a Master Cell Bank

To initiate the generation of a master cell bank (MCB) cells from one ormore of the preMCBs described above (see, Examples 2-4) are biologicallycloned through limiting dilution in order to ensure that the productioncells are derived from a unique genetic constellation. Clones are thenscreened for various phenotypic properties including doubling time andrelative tumorigenicity, as well as viral production. In an initialproof of concept experiment, fifty-four MDCK clones were obtained inmedia containing FCS. These clones were passaged and each was infectedwith a low multiplicity of infection of ca A/New Calcdonia/20/99.Several days after infection, the supernatant was removed and thequantity of virus in the supernatant was measured by TCID₅₀. A minorityof the clones produced relatively high titers of virus, greater than wasproduced in the noncloned parental cells. Clones with superiorbiological and physiological properties are used to establish a MasterCell Bank (MCB).

The MCB is extensively tested to ensure that there is no evidence ofadventitious agents. For example, one or more of several PCR and/orantibody-specific tests for available viral agents are conducted, asshown in Table 6, below.

TABLE 6 Testing Regimen For a MCB General tests PCR*/Ab specificSterility AAV Types 1 & 2 Mycoplasma HCMV Adventitious agents in vitro(multiple cell lines) EBV Adventitious agents in vivo HSV PERT HepatitisB, C & E Co-cultivation HHV 6, 7 & 8 Karyology HIV 1 & 2 Electronmicroscopy HPV Tumorigenicity intact cells (TP₅₀) HTLV I & IIOncogenicity of cellular DNA Polyoma (BK and Oncogenicity of cellularlysate JC viruses) Bovine viruses per 9CFR Circovirus Porcine virusesper 9CFR Canine Parvovirus Canine distemper Adenovirus SV40

Example 7 Process and Formulation of Vaccine Material

Use of a highly scalable microcarrier technology, similar to that usedfor the production of the currently licensed Polio vaccine, isapplicable to the production of influenza in MDCK cells. Spherical beadsmade of dextran support excellent growth of MDCK cells and in 2 to 10 Lbioreactors. Parental MDCK cells grown in SFMV 103 were found to becapable of growing on Cytodex 1 microcarriers to a density of 2×10⁶nuclei per mL in batch mode in both spinner flasks and MDCK cells havebeen grown to >1×10⁶ cell/mL in bioreactors up to a 10 L scale (data notshown). Initial pilot scale runs demonstrate that these MDCK cells arecapable of producing vaccine influenza strains to high titer in aserum-free process and the titers were found to be equivalent or greaterthan the productivity obtained using serum grown cells in T-flasks. Asshown in FIG. 20A, MDCK cells grown in Cytodex beads in 250 mL spinnerflasks produced high titers of H1N1, H3N2 and B vaccine strains. Forclinical manufacturing influenza virus may be produced in MDCK cells atthe 20 L or 150 L scale, while commercial scale production may utilized2,500 L bioreactors. FIG. 20B outlines one process that may be used forcell culture scale up to commercial production levels. The working cellbank is first expanded sequentially from a T-75 flask to T-225 flasks to1 liter spinner flasks to a 20 liter then 300 liter bioreactors whichare finally expanded to a 2500 liter bioreactor. When the optimal celldensity is obtained the culture in inoculated with the master viralstrain. The virus is then bulk harvested from the culture supernatant.

The purification process for cell culture based influenza vaccines ismodeled on purification of egg-based influenza vaccines (see, e.g., PCTPublication WO 05/014862 and PCT Patent Application PCT/US05/035614filed Oct. 4, 2005). The purification of viral vaccine materials fromcells may include any or all of the following processes, homogenation,clarification centrifugation, ultrafiltration, adsorption on bariumsulfate and elution, tangential flow filtration, density gradientultracentrifugation, chromatography, and sterialization filtration.Other purification steps may also be included. For example, crude mediumfrom infected cultures can first be clarified by centrifugation at,e.g., 1000-2000×g for a time sufficient to remove cell debris and otherlarge particulate matter, e.g., between 10 and 30 minutes.Alternatively, the medium is filtered through a 0.8 μm cellulose acetatefilter to remove intact cells and other large particulate matter.Optionally, the clarified medium supernatant is then centrifuged topellet the influenza viruses, e.g., at 15,000×g, for approximately 3-5hours. Following resuspension of the virus pellet in an appropriatebuffer, such as STE (0.01 M Tris-HCl; 0.15 M NaCl; 0.0001 M EDTA) orphosphate buffered saline (PBS) at pH 7.4, the virus may be concentratedby density gradient centrifugation on sucrose (60%-12%) or potassiumtartrate (50%-10%). Either continuous or step gradients, e.g., a sucrosegradient between 12% and 60% in four 12% steps, are suitable. Thegradients are centrifuged at a speed, and for a time, sufficient for theviruses to concentrate into a visible band for recovery. Alternatively,and for most large scale commercial applications, virus is elutriatedfrom density gradients using a zonal-centrifuge rotor operating incontinuous mode.

A feature which may included in the purification of viral vaccinematerials from cells is the use of Benzonase®, a non-specificendonuclease, early in the process. While MDCK cellular DNA does notpose an oncogenic risk based on studies evaluating oncogenicity ofcellular DNA, Benzonase® treatment would virtually eliminate anypotential or hypothetical risk. In one purification process, followingBenzonase® treatment, the material is clarified by direct flowfiltration (DFF) which will also remove any residual intact mammaliancells in the bulk material. The filtered bulk is then concentrated bytangential flow filtration (TFF) prior to further purification steps.Purification methods including affinity chromatography as well asion-exchange chromatography and/or hydroxyapatite which, have workedwell for other viral systems are useful for cell culture based influenzavaccine production. The highly purified viral material obtained by theprocess developed is then utilized in the production of vaccinematerial. For example, for use in a live attenuated vaccine production(e.g., FluMist®) the viral material may be subjected to a bufferexchange by filtration into a final formulation followed by asterilization step. Buffers useful for such a formulation may contain200 mM sucrose and a phosphate or histidine buffer of pH 7.0-7.2 withthe addition of other amino acid excipients such as arginine. Ifnecessary for stabilization protein hydrolysates such as porcine gelatinmay also be added. Ideally the vaccine material is formulated to bestable for an extended storage time. One method which may be utilized toextend storage time is spray drying, a rapid drying process whereby theformulation liquid feed is spray atomized into fine droplets under astream of dry heated gas. The evaporation of the fine droplets resultsin dry powders composed of the dissolved solutes (see, e.g., US PatentPublication 2004/0042972). Spray drying offers the advantages of ease ofscalability and manufacturing cost as compared to conventionalfreeze-drying processes. Alternatively, the vaccine material isformulated to be stable as a refrigerator stable liquid formulationusing methods known in the art. For example, methods and compositionsfor formulating a refrigerator stable attenuated influenza vaccine aredescribed in PCT Patent Application PCT/US2005/035614 filed Oct. 4,2005.

In-process characterization steps are incorporated into the purificationscheme to monitor the production. Characterization steps which may beutilized include but are not limited to Fluorescent Focus Assay (FFA,see, e.g., above) which uses a simple antibody binding and fluorescentstaining method to determine virus infectivity. Total protein and DNAdetermination which may be performed using numerous methods known to oneof skill in the art are used to determine the percent of the initialimpurities remaining. The specific activity of the preparation may bedetermined by calculating the viral infectivity per quantity of vaccine(e.g., infectivity/mg).

Example 8 Preclinical Animal Models

The ferret is a robust animal model used to evaluate the attenuation andimmunogenicity of attenuated influenza vaccines and component vaccinestrains. The performance of cell derived influenza strains produced fromthe MCB are compared to the same strains produced in eggs. Head to headcomparison of these materials in controlled studies enables a high levelof assurance of the comparability of these viral products.

In order to evaluate the ability of the two vaccines to infect orachieve a “take” in the ferret, animals are lightly anesthetized andinoculated intranasally with either the cell or egg produced viralpreparations. Nasal wash material is collected at several time pointsfollowing inoculation and the quantity of virus is evaluated by one ofseveral available methods in order to evaluate the kinetics and extentof viral replication in the animals' upper respiratory tract.Experiments are performed with a range of doses and include multiplestrains and different trivalent mixtures to generalize the relativeinfectivity of cell culture grown strains to egg produced strains. Thesesame studies are also used to evaluate the immunogenicity of theinfluenza strains, a property that is inherently linked to the abilityof the virus to initiate infection. Animals are bled and nasal washesare harvested at various points (weeks) post inoculation; thesespecimens are used to assess the serum antibody and nasal IgA responsesto infection. The culmination of these data, infectivity, serum antibodyand mucosal antibody responses, will be used to compare and evaluate therelative infectivity of the cell-produced vaccine to the egg producedvaccine. The most likely outcome is predicted to be that the cell andegg produced vaccine strains have similar infectivity andimmunogenicity. If the cell derived vaccine appeared to be moreinfective or more immunogenic than the egg-derived product, furtherstudies evaluating the possibility of lower dosage are performed.

A number of immunogenicity and replication studies are performed in theferret model to evaluate the cell culture-derived vaccines with a singleunit human dose. Infection with ca/ts/att strains generally elicitsstrong and rapid antibody responses in ferrets. In addition, individualca/ts/att strains are routinely tested and shown to express theattenuated (att) phenotype by replicating to relatively high titers inthe nasopharynx but to undetectable levels in the lung of these animals.The impact of cell culture growth on these biological traits is alsoassessed. However, it is unlikely that any differences will be seen,since the att phenotype is an integral part of the genetic compositionof these strains. The growth kinetics and crossreactivity of thesestrains is evaluated following administration of a single human dose inthese animals. Live attenuated vaccines generated from egg derivedmaterial elicit serum antibodies that cross-react with multiple strainswithin a genetic lineage; and it is expected that a cell-derived vaccinewill have the same capability.

These comparability evaluations should provide significant insight intopotential biochemical and/or biophysical differences of the primaryvirus product and demonstrate the impact of these epigenetic differenceson the performance of the ca/ts/att strains measured by first passagingthe virus in human cells or animal studies. Based on the sequenceinformation to date, there is no expected impact on the ca/ts/attstrains immunogenic performance resulting from production on MDCK cells.

Ferrets are a well document animal model for influenza and are usedroutinely to evaluate the attenuation phenotype and immunogenicity ofca/ts/att strains. In general, 8-10 week old animals are used to assessattenuation; typically study designs evaluate n=3-5 animals per test orcontrol group. Immunogenicity studies are evaluated in animals from 8weeks to 6 months of age and generally require n=3-5 animals per testarticle or control group. These numbers provide sufficient informationto obtain statistically valid or observationally important comparisonsbetween groups. During most studies Influenza-like signs may be noticed,but are not likely. Ferrets do not display signs of decrease in appetiteor weight, nasal or ocular discharge; observing signs of influenza-likeillness is a necessary part of the study and interventions such asanalgesics are not warranted. Other signs of discomfort, such as opensores or significant weight loss, would result in appropriatedisposition of the animal following discussion with the attendingveterinarian.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations. For example, all the techniques and apparatusdescribed above may be used in various combinations. All publications,patents, patent applications, or other documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication, patent,patent application, or other document were individually indicated to beincorporated by reference for all purposes.

The invention claimed is:
 1. A cell culture composition comprisingnon-tumorigenic and adherent Madin Darby Canine Kidney (MDCK) cells thathave been adapted to grow in a serum-free medium comprising Taub'smedium, lipids and wheat hydrolysate, which Taub's medium comprises a50:50 mixture of DMEM and Ham's F12, 4.5 grams per liter glucose, 4mML-glutamine, 5 micrograms per milliliter insulin, 5 micrograms permilliliter transferrin, 5picomolar triiodothyronine, 50 nanomolarhydrocortisone, 25 nanograms per milliliter prostaglandin El and 10nanomolar sodium selenite.
 2. The cell culture composition of claim 1,wherein the MDCK cells have been passaged at least 20 passages.
 3. Thecell culture composition of claim 1, wherein the MDCK cells are from thecell line MDCK-S deposited as ATCC Accession No. PTA-6500 or are fromthe cell line deposited as ATCC Accession No. CCL34.
 4. The cell culturecomposition of claim 1, wherein the MDCK cells are from the cell lineMDCK-SF103 deposited as ATCC Accession No. PTA-6503.
 5. The cell culturecomposition of claim 1, wherein the serum-free medium is MediV SF103. 6.The cell culture composition of claim 1, wherein the serum-free mediumfurther comprises epidermal growth factor (EGF).
 7. The cell culturecomposition of claim 6, wherein the MDCK cells are from the cell lineMDCK-S deposited as ATCC Accession No. PTA-6500 or are from the cellline deposited as ATCC Accession No. CCL34.
 8. The cell culturecomposition of claim 6, wherein the MDCK cells are from the cell lineMDCK-SF103 deposited as ATCC Accession No. PTA-6503.
 9. The cell culturecomposition of claim 6, wherein the serum-free medium is MediV SF104 orMediV SF105.
 10. The cell culture composition of claim 6, wherein theserum-free medium further comprises tropolone and lacks transferin. 11.The cell culture composition of claim 10, wherein the MDCK cells arefrom the cell line MDCK-S deposited as ATCC Accession No. PTA-6500 orare from the cell line deposited as ATCC Accession No. CCL34.
 12. Thecell culture composition of claim 10, wherein the MDCK cells are fromthe cell line MDCK-SF103 deposited as ATCC Accession No. PTA-6503. 13.The cell culture composition of claim 10, wherein the serum-free mediumis MediV SF105.
 14. The cell culture composition of claim 2, wherein theMDCK cells have been passaged at least 30 passages.
 15. The cell culturecomposition of claim 14, wherein the MDCK cells have been passaged atleast 40 passages.
 16. The cell culture composition of claim 15, whereinthe MDCK cells have been passaged at least 50 passages.
 17. The cellculture composition of claim 16, wherein the MDCK cells have beenpassaged at least 60 passages.
 18. An adherent, non-tumorigenic MadinDarby Canine Kidney (MDCK) cell line that can be cultivated inserum-free media and that can be infected by influenza viruses, which isprepared by a process comprising: (a) adapting MDCK cells to grow in aserum-free medium comprising Taub's medium, lipids and wheathydrolysate, which Taub's medium comprises a 50:50mixture of DMEM andHam's F12, 4.5 grams per liter glucose, 4 mM L-glutamine, 5microgramsper milliliter insulin, 5 micrograms per milliliter transferrin, 5picomolar triiodothyronine, 50 nanomolar hydrocortisone, 25 nanogramsper milliliter prostaglandin E1and 10 nanomolar sodium selenite; and (b)establishing a cell bank.
 19. The adherent, non-tumorigenic MDCK cellline of claim 18, wherein the MDCK cells are from the cell line MDCK-Sdeposited as ATCC Accession No. PTA-6500 or are from the cell linedeposited as ATCC Accession No. CCL34.
 20. The adherent, non-tumorigenicMDCK cell line of claim 18, wherein the MDCK cells are from the cellline MDCK-SF103 deposited as ATCC Accession No. PTA-6503.
 21. Theadherent, non-tumorigenic MDCK cell line of claim 18, wherein theserum-free medium is MediV SF103.
 22. The adherent, non-tumorigenic MDCKcell line of claim 18, wherein the serum-free medium further comprisesepidermal growth factor (EGF).
 23. The adherent, non-tumorigenic MDCKcell line of claim 22, wherein the MDCK cells are from the cell lineMDCK-S deposited as ATCC Accession No. PTA-6500 or are from the cellline deposited as ATCC Accession No. CCL34.
 24. The adherent,non-tumorigenic MDCK cell line of claim 22, wherein the MDCK cells arefrom the cell line MDCK-SF103 deposited as ATCC Accession No. PTA-6503.25. The adherent, non-tumorigenic MDCK cell line of claim 22, whereinthe serum-free medium is MediV SF104 or MediV SF105.
 26. The adherent,non-tumorigenic MDCK cell line of claim 22, wherein the serum-freemedium further comprises tropolone and lacks transferin.
 27. Theadherent, non-tumorigenic MDCK cell line of claim 26, wherein the MDCKcells are from the cell line MDCK-S deposited as ATCC Accession No.PTA-6500 or are from the cell line deposited as ATCC Accession No.CCL34.
 28. The adherent, non-tumorigenic MDCK cell line of claim 26,wherein the MDCK cells are from the cell line MDCK-SF103 deposited asATCC Accession No. PTA-6503.
 29. The adherent, non-tumorigenic MDCK cellline of claim 26, wherein the serum-free medium is MediV SF105.
 30. Theadherent, non-tumorigenic MDCK cell line of claim 18, wherein the MDCKcells have been passaged at least 20 passages.
 31. The adherent,non-tumorigenic MDCK cell line of claim 30, wherein the MDCK cells havebeen passaged at least 30 passages.
 32. The adherent, non-tumorigenicMDCK cell line of claim 31, wherein the MDCK cells have been passaged atleast 40 passages.
 33. The adherent, non-tumorigenic MDCK cell line ofclaim 32, wherein the MDCK cells have been passaged at least 50passages.
 34. The adherent, non-tumorigenic MDCK cell line of claim 33,wherein the MDCK cells have been passaged at least 60 passages.
 35. Theadherent, non-tumorigenic MDCK cell line of claim 22, wherein theserum-free medium is MediV SF104.
 36. The cell culture composition ofclaim 6, wherein the serum-free medium is MediV SF104.